Composition of lactoferrin related peptides and uses thereof

ABSTRACT

The present invention is directed to a composition consisting of a series of novel biologically active 33-mer peptides.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.60/622,176 filed Oct. 26, 2004, which is incorporated herein byreference in its entirety.

TECHNICAL FIELD

The present invention is directed to a composition consisting of aseries of novel biologically active 33-mer peptides.

BACKGROUND OF THE INVENTION

Lactoferrin is a single chain metal binding glycoprotein. Many celltypes, such as monocytes, macrophages, lymphocytes, and brush-bordercells in the intestine, are known to have lactoferrin receptors.Lactoferrin is found mainly in external secretions of mucosal epitheliasuch as breast milk, saliva, tears, bile, and pancreatic fluid and has awide array of functions related to host primary defense mechanisms. Forexample, lactoferrin has been reported to activate natural killer (NK)cells, induce colony stimulating activity, activate polymorphonuclearneutrophils (PMN), regulate granulopoeisis, enhance antibody-dependentcell cytotoxicity, stimulate lymphokine-activated killer (LAK) cellactivity, and potentiate macrophage toxicity. Lactoferrins have beenreported to have a number of beneficial effects includinganti-infective, anti-inflammatory, immunomodulatory and anti-canceractivities.

Recombinant human lactoferrin has previously been described as beingpurified after expression in a variety of prokaryotic and eukaryoticorganisms including aspergillus (U.S. Pat. No. 6,080,559), cattle (U.S.Pat. No. 5,919,913), rice, corn, Sacharomcyes (U.S. Pat. No. 6,228,614)and Pichia pastoris (U.S. Pat. Nos. 6,455,687, 6,277,817, 6,066,469).Also described are expression systems for the expression of full-lengthhuman lactoferrins (e.g. U.S. Pat. No. 6,100,054). In all cases, part ofthe teaching is expression of the full length cDNA and purification ofthe intact protein whose N-terminal, after processing of the leaderpeptide, is the amino acid glycine. Nuijens, et al. (U.S. Pat. No.6,333,311) separately describe variants of human lactoferrin but theirfocus is limited to deletion or substitution of arginine residues foundin the N-terminal domain of lactoferrin.

LF contains a strongly basic region close to its N-terminus and binds toa variety of anionic biological molecules including lipid A (Appelmelket al., Infect. Immun. 62: 2628-2632 (1994)) and glycosaminoglycanswhich occur on the surface of most cells and in most extracellularmatrices (Mann et al., J. Biol. Chem. 269: 23661-7 (1994)).Lactoferricin H (residues 1-47) and lactoferricin B (residue 17-41) arereleased by pepsinolysis of human or bovine LF, respectively, and mayhave more potent antibacterial activity than the native proteins(Bellamy et al., Biochim. Biophys. Acta. 1121: 130-136 (1992)). A regioncomposed of residues 28-34 is reported to contribute to the highaffinity binding of human LF and lactoferricin H to endotoxin(Elass-Rochard et al., Biochem. J. 312: 839-845 (1995)). LF andlactoferricin B have been shown to inhibit the endotoxin-inducedinterleukin-6 response in human monocytic cells (Mattsby-Baltzer et al.,Pediatr. Res. 40: 257-262 (1996)). Previously identified fragments of LFwhich exhibit antimicrobial activity were isolated from pepsinhydrolysates of LF (Tomita et al., (1993) U.S. Pat. No. 5,214,028;Tomita et al., (1994) U.S. Pat. No. 5,304,633; Tomita et al., (1994)U.S. Pat. No. 5,317,084; Tomita et al., (1997) U.S. Pat. No. 5,656,591).

Previous studies have established that the N-terminal 33 residues ofhuman LF represent the minimal sequence that mediates binding of theprotein to anionic polysaccharides such as glycosaminoglycans (Mann etal., J. Biol. Chem. 269: 23661-7 (1994)). This sequence contains acationic head (residues 1-6) and tail (residues 28-33) which combine toform the glycosaminoglycan-binding site. In further studies described byMann in U.S. Pat. No. 6,399,570, lactoferrin peptide fragments having upto the first 51 amino acids encompassed endotoxin neutralizing abilityand antimicrobial activity. Mann further showed that up to 17 aminoacids could be deleted from the C-terminus and up to 3 amino acids couldbe deleted N-terminus of the 51 amino acid long peptide while retainingthe endotoxin neutralizing ability and antimicrobial activity.

The present invention is the first to describe a peptide compositionthat consists of lactoferrin related peptides which are at least 33amino acids in length in which at least four amino acids at the C and/orN terminus are substituted for positive amino acids resulting in anenhancement of its biological activity.

BRIEF SUMMARY OF THE INVENTION

The present invention is directed to a composition consisting of aseries of biologically active peptides. The peptides of the presentinvention comprise at least 33 amino acids in which at least four aminoacids at the C and/or N terminus are substituted for positive aminoacids. These biologically active peptides can be used to treat a varietyof pathological conditions, for example, but not limited tohyperproliferative disease, respiratory disorder, cardiovasculardisease, neurological condition, autoimmune disorder, infectiousdisease, gastrointestinal disorder, endocrine and/or metabolismdisorder, hematological disorder, ocular disorder, integument disorder,pain and a wound.

One embodiment of the present invention comprises a pharmaceuticalcomposition that induces modulation of the immune system whereby thecomposition stimulates production of MIP-3alpha from hepatocytes. Thecomposition can also inhibit bacterial growth as measured by minimuminhibitory concentration (MIC). More specifically, the compositioncomprises an amino acid sequence selected from the group consisting ofSEQ. ID. NO.1, SEQ. ID. NO.2, SEQ. ID. NO.3, SEQ. ID. NO.4, SEQ. ID.NO.5, SEQ. ID. NO.6, SEQ. ID. NO.7, SEQ. ID. NO.8, SEQ. ID. NO.9, SEQ.ID. NO.10, and SEQ. ID. NO.11.

In certain embodiments, the pharmaceutical composition comprises atleast four consecutive positively charged residues at the N-terminus orat least four positively charged residues at the C-terminus or at leastfour positively charged residues at each of the N- and C-termini. Thus,the pharmaceutical composition may comprise at least 25% positivelycharged residues or at least 33% positively charged residues. Thepositively charged residues are arginine, lysine or derivates thereof.

In further embodiments, the pharmaceutical composition can furthercomprise a metal chelator. The metal chelator is selected from the groupconsisting of ethylenediaminetetraacetic acid (EDTA),[ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA),1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA), andhydroxyethlene triamine diacetic acid, (HEDTA).

Another embodiment of the present invention comprises an isolatedpolypeptide having an amino acid sequence as defined in SEQ. ID. NO. 1,SEQ. ID. NO.2, SEQ. ID. NO.3, SEQ. ID. NO.4, SEQ. ID. NO.5, SEQ. ID.NO.6, SEQ. ID. NO.7, SEQ. ID. NO.8, SEQ. ID. NO.9, SEQ. ID. NO.10, orSEQ. ID. NO.11. The polypeptide is admixed with a pharmaceuticallyacceptable carrier.

Yet further, another embodiment comprises an expression vectorcomprising a nucleic acid sequence encoding an amino acid sequenceselected from the group consisting of SEQ. ID. NO. 1, SEQ. ID. NO.2,SEQ. ID. NO.3, SEQ. ID. NO.4, SEQ. ID. NO.5, SEQ. ID. NO.6, SEQ. ID.NO.7, SEQ. ID. NO.8, SEQ. ID. NO.9, SEQ. ID. NO.10, and SEQ. ID. NO.11.The expression vector is further defined as a viral or plasmid vector.The viral vector is an adenoviral vector, an adeno-associated viralvector, a retroviral vector, a lentiviral vector, a herpes viral vector,polyoma viral vector, pox vector, or hepatitis B viral vector.

In certain embodiments, the present invention comprises a method oftreating a pathological condition comprising the step of administeringthe pharmaceutical composition, or the polypeptide or the expressionvector. It is understood that the pharmaceutical composition or thepolypeptide or the expression vector can be combined with a standardtherapy to treat the pathological condition.

The pathological condition is selected from the group consisting ofhyperproliferative disease, respiratory disorder, cardiovasculardisease, neurological condition, autoimmune disorder, infectiousdisease, gastrointestinal disorder, endocrine and/or metabolismdisorder, hematological disorder, ocular disorder, integument disorder,pain and a wound.

More specifically, the hyperproliferative disease is further defined ascancer. Exemplary cancers include, but are not limited to melanoma,non-small cell lung cancer, small-cell lung cancer, lung cancer,leukemia, hepatocarcinoma, retinoblastoma, astrocytoma, glioblastoma,gum cancer, tongue cancer, neuroblastoma, head cancer, neck cancer,breast cancer, pancreatic cancer, prostate cancer, renal cancer, bonecancer, testicular cancer, ovarian cancer, mesothelioma, cervicalcancer, gastrointestinal cancer, lymphoma, myeloma, brain cancer, coloncancer, sarcoma or bladder cancer.

In certain embodiments the pharmaceutical composition or the polypeptideor the expression vector can be combined with a standard anti-cancertherapy to treat the cancer. The anti-cancer agent selected from thegroup consisting of chemotherapy, surgery, radiotherapy, animmunostimulant, anti-cancer biologic agent or a cancer vaccine

Yet further, hyperproliferative disease can also encompass rheumatoidarthritis, infectious arthritis, inflammatory bowel disease,osteoarthritis, leiomyomas, adenomas, lipomas, hemangiomas, fibromas,vascular occlusion, restenosis, atherosclerosis, pre-neoplastic lesions,carcinoma in situ, oral hairy leukoplakia, or psoriasis.

Exemplary neurological conditions that may be treated using thepharmaceutical composition or the polypeptide or the expression vectorof the present invention can include, but are not limited to multiplesclerosis, Alzheimer's disease, Parkinson's disease, muscular dystrophy,sleep or depression.

Gastrointestinal disorders that can be treated using the pharmaceuticalcomposition or the polypeptide or the expression vector of the presentinvention can include, but are not limited to Crohn's disease, colitis,necrotizing enterocolitis, endometriosis, irritable bowel syndrome,pancreatitis, periodontal disease, and ulcerative colitis.

Exemplary autoimmune disorders that can be treated using thepharmaceutical composition or the polypeptide or the expression vectorof the present invention can include, but are not limited to graftversus host disease (GVHD), organ transplant rejection, autoimmunehepatitis, primary biliary cirrhosis, autoimmune cholangitis, primarysclerosing cholangitis, irritable bowel syndrome (IBS), multiplesclerosis (MS), chronic granulomatous disease, ankylosing spondylitis,scleroderma, polymyositis, (dermato)myositis, systemic vasculitis,systemic lupus erythematosus (SLE), Chrohn's disease, insulin-dependentdiabetes (type 1) or ulcerative colitis.

Endocrine and/or metabolism disorder that can be treated using thepharmaceutical composition or the polypeptide or the expression vectorof the present invention can include, but are not limited to diabetesmellitus, thyroid disorder, or osteoporosis.

Hematological disorders that can be treated using the pharmaceuticalcomposition or the polypeptide or the expression vector of the presentinvention can include, but are not limited to anemia, sickle cell anemiaor cachexia.

Infectious diseases that can be treated using the pharmaceuticalcomposition or the polypeptide or the expression vector of the presentinvention can include, but are not limited to a disease that result frominfection of either a virus, a bacterium or a fungus or a combinationthereof. Exemplary viruses include, but are not limited to herpessimplex, labilis zoster, HIV, hepatitis A, hepatitis, B, or hepatitis C.Infectious disease can include HIV, hepatitis, West Nile Virus bacterialmeningitis, paramenigeal infections, septic thrombophlebitis,candidiasis, myocarditis, bacteremia, sepsis, or septic shock.

Ocular disorder that can be treated using the pharmaceutical compositionor the polypeptide or the expression vector of the present invention caninclude, but are not limited to conjunctivitis, dry eye disease,glaucoma, allergic eye disease, uveitis or ocular infection.

Cardiovascular disease that can be treated using the pharmaceuticalcomposition or the polypeptide or the expression vector of the presentinvention can include, but are not limited to congestive heart failure,hypertension, cardiomyopathy, myocarditis, atherosclerosis, chronicvenous disease, or heart arrhythmia.

In further embodiments, the pathological condition is elevatedcholesterol or dyslipidemia.

The wound that can be treated using the pharmaceutical composition orthe polypeptide or the expression vector of the present invention caninclude, but are not limited to skin wound, internal wound,gastrointestinal wound, oral wound, ophthalmic wound, surgical wound andfractures.

The respiratory disorder that can be treated using the pharmaceuticalcomposition or the polypeptide or the expression vector of the presentinvention can include, but are not limited to atopic asthma, non-atopicasthma, emphysema, bronchitis, chronic obstructive pulmonary disease(COPD), sinusitis, allergic rhinitis, fibrotic lung disease, ARDS,pulmonary vascular disease/pulmonary hypertension, Cor Pulmonale, orcystic fibrosis.

In further embodiments, the composition stimulates the production ofIL-18. Yet further, the composition stimulates the production of IL-12,GM-CSF, or IFN-γ. Still further, composition stimulates or supplementsimmune cells, exemplary immune cells are T lymphocytes, B-lymphocytes,dendritic cells (DCs), other antigen presenting cells (APCs), naturalkiller (NK) cells, macrophages or monocytes. The T lymphocytes areselected from the group consisting of CD4+, CD8+, CD3+ and CD40+ cellsand the B-lymphocytes are CD40+ cells.

In certain embodiments, the composition is administered orally,inhalation, nasally, topically or parenterally. Parenterally can includesubcutaneously, intramuscularly, intraperitoneally, intravenously,intraarterially, intramyocardially, transendocardially,transepicardially, intrathecally or intra-tumorally.

The foregoing has outlined rather broadly the features and technicaladvantages of the present invention in order that the detaileddescription of the invention that follows may be better understood.Additional features and advantages of the invention will be describedhereinafter which form the subject of the claims of the invention. Itshould be appreciated by those skilled in the art that the conceptionand specific embodiment disclosed may be readily utilized as a basis formodifying or designing other structures for carrying out the samepurposes of the present invention. It should also be realized by thoseskilled in the art that such equivalent constructions do not depart fromthe spirit and scope of the invention as set forth in the appendedclaims. The novel features which are believed to be characteristic ofthe invention, both as to its organization and method of operation,together with further objects and advantages will be better understoodfrom the following description when considered in connection with theaccompanying figures. It is to be expressly understood, however, thateach of the figures is provided for the purpose of illustration anddescription only and is not intended as a definition of the limits ofthe present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the present invention, reference isnow made to the following descriptions taken in conjunction with theaccompanying drawing, in which:

FIG. 1 shows in vitro NK cell activation, as measured by the percentkilling of target cells, by peptide LPF-35.

FIG. 2 shows T cell stimulation by peptide LPF-35 for various cellsurface cluster markers including CD3/CD4, CD3/CD8, CD4/CD69 andCD8/CD69.

FIG. 3 shows the reduction in mortality achieved with peptide LPF-35 ina mouse model of sepsis versus placebo and a positive control usingrecombinant human lactoferrin.

FIG. 4 shows the reduction in eye damage achieved using variousconcentrations of peptide LPF-35 versus saline control in a rabbit modelof dry eye disease.

FIGS. 5A and 5B show the effects of a single oral dose of either peptideLPF-32 or peptide LPF-35 on lung resistance (FIG. 5A) and airwayhyperresponsiveness (FIG. 5B) to carbachol in a sheep model of asthma.

FIGS. 6A and 6B show the effects of peptide LPF-35, at various dosesafter 3 days of oral administration, on lung resistance (FIG. 6A) andairway hyperresponsiveness (FIG. 6B) to carbachol in a sheep model ofasthma.

FIGS. 7A and 7B show that the rate and incidence of wound healing areincreased following topical treatment with different concentrations ofpeptide LPF-35 in a mouse model of chronic wounds. FIG. 7A shows theeffects on acceleration of wound closure. FIG. 7B shows the increase inincidence of wound healing.

FIGS. 8A and 8B show the effect of oral administration of peptideLPF-35, at different doses, on decreasing the blood levels of glucose(FIG. 8A) and insulin (FIG. 8B) in a mouse model of diabetes.

FIG. 9 shows the effect of peptide LPF-35, at different doses and timepoints, on the expression of MIP-3-alpha in hepatocytes.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. For purposes of the presentinvention, the following terms are defined below.

As used herein, the use of the word “a” or “an” when used in conjunctionwith the term “comprising” in the claims and/or the specification maymean “one,” but it is also consistent with the meaning of “one or more,”“at least one,” and “one or more than one.” Still further, the terms“having”, “containing”, “including” and “comprising” are interchangeableand one of skill in the art is cognizant that these terms are open endedterms.

The term “acute pain” as used herein includes tissue injury orpathology. For example, acute pain includes, but is not limited to painfollowing surgery (e.g., surgery), pain following trauma (e.g., blunt orsharp injury, bullet wounds), pain associated with athletic injury, andthe occasional headache. Medically, acute pain can have a diagnosticvalue; that is, it leads to the discovery of a pathological condition.Yet further, acute pain lasts or is anticipated to last for a shortperiod of time, typically less than one to two months. Acute pain isassociated with hyperactivity of the sympathetic nervous system, forexample, tachycardia, increased respiratory rate, increased bloodpressure, increased cortisol release, diaphoresis and dilated pupils.

The term “antibiotic” as used herein is defined as a substance thatinhibits the growth of microorganisms without damage to the host. Forexample, the antibiotic may inhibit cell wall synthesis, proteinsynthesis, nucleic acid synthesis, or alter cell membrane function.Classes of antibiotics that can possibly be used include, but are notlimited to, macrolides (e.g., erythromycin), penicillins (e.g.,nafcillin), cephalosporins (e.g., cefazolin), carbepenems (e.g.,imipenem, aztreonam), other beta-lactam antibiotics, beta-lactaminhibitors (e.g., sulbactam), oxalines (i.e. linezolid), aminoglycosides(e.g., gentamicin), chloramphenicol, sulfonamides (e.g.,sulfamethoxazole), glycopeptides (e.g., vancomycin), quinolones (e.g.,ciprofloxacin), tetracyclines (e.g., minocycline), fusidic acid,trimethoprim, metronidazole, clindamycin, mupirocin, rifamycins (e.g.,rifampin), streptogramins (e.g., quinupristin and dalfopristin)lipoprotein (e.g., daptomycin), polyenes (e.g., amphotericin B), azoles(e.g., fluconazole), and echinocandins (e.g., caspofungin acetate). Theterm “morbidity” as used herein is the state or condition of beingdiseased. Yet further, morbidity can also refer to the ratio ofincidence, for example the number of sick subjects or cases of diseasesin relationship to a specific population.

The term “anti-depressant drug” as used herein includes compounds thatprovide pain relief by reducing side-effects including anxiety.Anti-depressant drugs include, for example, but are not limited totricyclin antidepressants, sedatives, tranquilizers, hypnotics,anti-histamines, and amphetamines.

The term “antimicrobial” as used herein is defined as a substance thatinhibits the growth of microorganisms without damage to the host, forexample antibiotics, anti-fungal and antiseptics.

The term “atherosclerosis” as used herein includes a form ofarteriosclerosis characterized by a combination of changes in the intimaof arteries, such changes include, but are not limited to accumulationof lipids, complex carbohydrates, blood and blood products, fibroustissue and calcium deposits. Yet further, atherosclerotic plaques can becharacterized into at least two areas. One type is characterized byprominent proliferation of cells with small accumulations of lipids. Thesecond type consists mostly of intracellular and extracellular lipidaccumulation and a small amount of cellular proliferation.

The term “bacteremia” as used herein is defined as having a focus ofbacterial infection or bacteria in the blood of the subject.

The term “cancer” as used herein is defined as a hyperproliferation ofcells whose unique trait—loss of normal control—results in unregulatedgrowth, lack of differentiation, local tissue invasion, and metastasis.Examples include but are not limited to, melanoma, non-small cell lung,small-cell lung, lung, hepatocarcinoma, leukemia, retinoblastoma,astrocytoma, glioblastoma, gum, tongue, neuroblastoma, head, neck,breast, pancreatic, prostate, renal, bone, testicular, ovarian,mesothelioma, cervical, gastrointestinal, lymphoma, brain, colon,sarcoma or bladder cancer.

The term “cardiovascular disease or disorder” as used herein refers todisease and disorders related to the cardiovascular or circulatorysystem. Cardiovascular disease and/or disorders include, but are notlimited to, diseases and/or disorders of the pericardium, heart valves(e.g., incompetent valves, stenosed valves, rheumatic heart disease,mitral valve prolapse, aortic regurgitation), myocardium (e.g., coronaryartery disease, myocardial infarction, heart failure, ischemic heartdisease, angina), blood vessels (e.g., hypertension, arteriosclerosis,aneurysm) or veins (e.g., varicose veins, hemorrhoids). Yet further, oneskilled in the art recognizes that cardiovascular diseases and/ordisorders can result from congenital defects, genetic defects,environmental influences (e.g., dietary influences, lifestyle, stress,etc.), and other defects or influences.

The term “cell,” “cell line,” and “cell culture” as used herein may beused interchangeably. All of these terms also include their progeny,which are any and all subsequent generations. It is understood that allprogeny may not be identical due to deliberate or inadvertent mutations.

The term “chemokine” as used herein refers to small cytokines that areinvolved in the migration and activation of cells, for examplephagocytic cells and lymphocytes. One of skill in the art realizes thatchemokines play a central role in inflammatory and immune responseprocesses.

The term “cholesterol” as used herein refers to the monohydric alcoholform, which is a white, powdery substance that is found in all animalcells and in animal-based foods (not in plants). Cholesterol is anessential nutrient necessary for many functions, including thefollowing: repairing cell membranes, manufacturing vitamin D on theskin's surface, production of hormones, such as estrogen andtestosterone, and possibly helping cell connections in the brain thatare important for learning and memory.

The term “chronic pain” as used herein refers to pain that lasts longerthan 1 month or beyond the resolution of an acute tissue injury or isrecurring or is associated with tissue injury and/or chronic diseasesthat are expected to continue or progress, for example, cancer,arthritis, inflammatory disease, chronic wounds, cardiovascularaccidents, spinal cord disorders, central nervous system disorder orrecovery from surgery. Chronic pain can be associated with severalfactors that include, but are not limited to lassitude, sleepdisturbance, decreased appetite, loss of taste for food, weight loss,diminished libido, constipation, or depression.

The term “chylomicron” as used herein refers to the largest in size andlowest in density of the triglyceride carrying lipoproteins.

The term “cytokine” as used herein refers to proteins that are made bycells that affect the behavior of other cells, for example stimulate orinhibit cell proliferation. For example, cytokines that are made bylymphocytes are often called lymphokines or interleukins. One of skillin the art realizes that the term cytokine is a generic term used in theliterature to refer to proteins that are made by cells that can affectthe behavior of other cells.

The term “diabetes mellitus” as used herein refers to a disorder ofcarbohydrate metabolism that is typically characterized by hyperglycemiaand glycosuria, which results from inadequate production or utilizationof insulin. Diabetes mellitus includes several syndromes or disorders,for example, but not limited to, primary diabetes mellitus (e.g.,insulin-dependent (Type I) and non-insulin-dependent (Type II));secondary diabetes [for example: pancreatic diabetes (e.g., destructionof the pancreas, removal of the pancreas, etc.);extrapancreatic/endocrine diabetes (e.g., hypersomatotropism,hyperadrenalism, hyperthyroidism, glucagonama, etc.); drug-induceddiabetes (e.g., steroid diabetes, thiazides, etc.)] and rare/exceptionalforms of diabetes (e.g., lipoatrophic diabetes, myatonic diabetes,disturbance of insulin receptors, genetic syndromes, etc). Long-termcomplications of diabetes include neuropathy, retinopathy, nephropathy,generalized degenerative changes in the blood vessels and increasedsusceptibility to infection.

The term “effective amount” or “therapeutically effective amount” asused herein refers to an amount that results in an improvement orremediation of the symptoms of the disease or condition.

The term “expression construct” or “transgene” is defined as any type ofgenetic construct containing a nucleic acid coding for gene products inwhich part or all of the nucleic acid encoding sequence is capable ofbeing transcribed can be inserted into the vector. The transcript istranslated into a protein, but it need not be. In certain embodiments,expression includes both transcription of a gene and translation of mRNAinto a gene product. In other embodiments, expression only includestranscription of the nucleic acid encoding genes of interest. In thepresent invention, the term “therapeutic construct” may also be used torefer to the expression construct or transgene.

The term “expression vector” refers to a vector containing a nucleicacid sequence coding for at least part of a gene product capable ofbeing transcribed. In some cases, RNA molecules are then translated intoa protein, polypeptide, or peptide. Expression vectors can contain avariety of control sequences, which refer to nucleic acid sequencesnecessary for the transcription and possibly translation of anoperatively linked coding sequence in a particular host organism. Inaddition to control sequences that govern transcription and translation,vectors and expression vectors may contain nucleic acid sequences thatserve other functions as well and are described infra. In the presentinvention, the term “therapeutic vector” may also be used to refer tothe expression vector.

The term “fragile bone condition” or “fragile bone disease” as usedherein refers to a condition or disease characterized by low bone massor structural deterioration of bone tissue, leading to bone fragilityand increased susceptibility to fractures.

The term “gastrointestinal disorder or conditions” as used hereinincludes gastrointestinal disorders in which one or more of the symptomsand conditions affect the gastrointestinal tract from the mouth to theanus, as well as any organ that may play a role in digestion, forexample, but not limited to the pancreas, or the gall bladder.Gastrointestinal disorders include, but are not limited to, heartburn,bloating, postoperative ileus, abdominal pain and discomfort, earlysatiety, epigastric pain, nausea, vomiting, burbulence, regurgitation,intestinal pseudoobstruction, anal incontinence, gastroesophageal refluxdisease, irritable bowel syndrome, ulcerative colitis, Crohn's disease,renal disorders, menstrual cramps, pancreatitis, spastic andinterstitial cystitis and ulcers and the visceral pain associatedtherewith.

The term “Grade 3 neutropenia” refers to the reduction of the absoluteneutrophil cell count (ANC) to less than about 1000 cells/μL. The term“neutropenia” generally refers to a condition in which the ANC isreduced to 1000 cells/μL or less. Such a condition may be caused bydepressed production, increased peripheral destruction of neutrophils.The most common neutropenias are iatrogenic, resulting from thewidespread use of cytotoxic or immunosuppressive therapies for cancertreatment or control of autoimmune disorders. Other causes ofneutropenia include induction by drugs, hematological diseases includingidiopathic, cyclic neutropenia, Chediak-Higashi syndrome, aplasticanemia, infantile genetic disorders, tumor invasion such asmyelofibrosis, nutritional deficiency; infections such as tuberculosis,typhoid fever, brucelloisis, tularemia, measles, infectiousmononucleosis, malaria, viral hepatitis, leishmaniasis, AIDS,antineutrophil antibodies and/or splenetic or lung trapping, autoimmunedisorders, wegner's granulomatosis, acute endotoxemia, hemodialysis, andcardiopulmonary bypass. The present invention applies to any acquiredand inherited neutropenic conditions.

The term “graft-versus-host-disease” or “GVHD” as used herein is thepathological reaction that occurs between the host and grafted tissue.The grafted or donor tissue dominates the pathological reaction.Graft-versus-host-disease (GVHD) can be seen following stem cell and/orsolid organ transplantation. GVHD occurs in immunocompromised subjects,who when transplanted, receive “passenger” lymphocytes in thetransplanted stem cells or solid organ. These lymphocytes recognize therecipient's tissue as foreign. Thus, they attack and mount aninflammatory and destructive response in the recipient. GVHD has apredilection for epithelial tissues, especially skin, liver, and mucosaof the gastrointestinal tract. GVHD subjects are immunocompromised dueto the fact that prior to transplant of the graft, the subject receivesimmunosuppressive therapy.

The term “gram-negative bacteria” or “gram-negative bacterium” as usedherein is defined as bacteria which have been classified by the Gramstain as having a red stain. Gram-negative bacteria have thin walledcell membranes consisting of a single layer of peptidoglycan and anouter layer of lipopolysaccharide, lipoprotein, and phospholipid.Exemplary organisms include, but are not limited to, Enterobacteriaceaconsisting of Escherichia, Shigella, Edwardsiella, Salmonella,Citrobacter, Klebsiella, Enterobacter, Hafnia, Serratia, Proteus,Morganella, Providencia, Yersinia, Erwinia, Buttlauxella, Cedecea,Ewingella, Kluyvera, Tatumella and Rahnella. Other exemplarygram-negative organisms not in the family Enterobacteriacea include, butare not limited to, Pseudomonas aeruginosa, Stenotrophomonasmaltophilia, Burkholderia, Cepacia, Gardenerella, Vaginalis, andAcinetobacter species.

The term “gram-positive bacteria” or “gram-positive bacterium” as usedherein refers to bacteria, which have been classified using the Gramstain as having a blue stain. Gram-positive bacteria have a thick cellmembrane consisting of multiple layers of peptidoglycan and an outsidelayer of teichoic acid. Exemplary organisms include, but are not limitedto, Staphylococcus aureus, coagulase-negative staphylococci,streptococci, enterococci, corynebacteria, and Bacillus species.

The term “high-density lipoprotein” or “HDL” as used herein is thesmallest and most dense type of cholesterol-carrying lipoprotein and isoften referred to as the “good” cholesterol.

The term “hyperproliferative disease” is defined as a disease thatresults from a hyperproliferation of cells. Exemplary hyperproliferativediseases include, but are not limited to cancer or autoimmune diseases.Other hyperproliferative diseases may include vascular occlusion,restenosis, atherosclerosis, or inflammatory bowel disease.

The term “immunocompromised” as used herein is defined as a subject whois, at the time of pathogen exposure, has a pre-existing condition thatreduces one or more mechanisms for normal defense against infection. Theimmunocompromised condition may be due to a defect or dysfunction of theimmune system or to other factors that heighten susceptibility toinfection, for example immunosuppressive agents. Although such acategorization allows a conceptual basis for evaluation,immunocompromised individuals with infection often do not fit completelyinto one group or the other. More than one defect in the body's defensemechanisms may be affected. For example, an immunocompromised state canresult from indwelling central lines or other types of impairment due tointravenous drug abuse; or be caused by secondary malignancy,malnutrition, or having been infected with other infectious agents suchas tuberculosis, influenza, Staphylococcus aureus or sexuallytransmitted diseases, e.g., syphilis or hepatitis.

The term “insulin-dependent diabetes mellitus”; “IDDM” or “Type I” referto diabetes that is characterized by a hyperglycemia, glycosuria, andlow blood insulin levels. Type I diabetes can develop at any age. Ittypically has an abrupt onset during the first two decades of life.Insulin therapy is usually required.

The term “intermediate density lipoprotein” or “IDL” as used hereinrefers to a triglyceride-carrying lipoprotein.

The term “lactoferrin composition” as used herein refers to acomposition having a lactoferrin related peptide.

The term “lactoferrin” or “LF” as used herein refers to native orrecombinant lactoferrin. Native lactoferrin can be obtained bypurification from mammalian milk or colostrum or from other naturalsources. Recombinant lactoferrin (rLF) can be made by recombinantexpression or direct production in genetically altered animals, plants,fungi, bacteria, or other prokaryotic or eukaryotic species, or throughchemical synthesis.

The term “lipid” as used herein refers to the building blocks of any ofthe fats or fatty substances found in animals and plants, which arecharacterized by their insolubility in water and solubility in fatsolvents such as alcohol, ether and chloroform. Lipids include fats(e.g., esters of fatty acids and glycerol); lipoids (e.g.,phospholipids, cerebrosides, waxes) and sterols (e.g., cholesterol).

The term “lipoproteins” as used herein are protein spheres thattransport cholesterol, triglyceride, or other lipid molecules throughthe bloodstream. Lipoproteins are categorized into five types accordingto size and density. They can be further defined by whether they carrycholesterol [the two smaller lipoproteins (HDL and LDL)] ortriglycerides [the three largest lipoproteins (IDL, VLDL, andchylomicrons)].

The term “low density lipoprotein” or “LDL” as used herein is a type ofcholesterol-carrying lipoprotein which is often called the “bad”cholesterol.

The term “metal chelator” as used herein refers to a compound whichbinds metal. Metal chelators that can be used in the present inventioninclude the divalent metal chelators, for example,ethylenediaminetetraacetic acid (EDTA),[ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA),1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA),hydroxyethlene triamine diacetic acid (HEDTA), or salts thereof.

The term “morbidity” as used herein is the state of being diseased. Yetfurther, morbidity can also refer to the disease rate or the ratio ofsick subjects or cases of disease in to a given population.

The term “mortality” as used herein is the state of being mortal orcausing death. Yet further, mortality can also refer to the death rateor the ratio of number of deaths to a given population.

The term “neurology” or “neurological” refers to conditions, disorders,and/or diseases that are associated with the nervous system. Thus, anycondition, disorder and/or disease that effects any component or aspectof the nervous system (either central or peripheral) is referred to as aneurological condition, disorder and/or disease. As used herein, theterm “neurological” or “neurology” encompasses the terms“neuropsychiatric” or “neuropsychiatry” and “neuropsychological” or“neuropsychological”. Thus, a neurological disease, condition, ordisorder includes, but is not limited to cognitive disorders, affectivedisorders (e.g., depression disorders and/or anxiety disorders),movement disorders, mental disorders, pain disorders, sleep disorders,etc.

The term “neutropenia” as used herein refers to an a decrease or smallnumber of neutrophils in the blood compared to normal. For example, theWorld Health Organization defines neutropenia as a subject having anabsolute neutrophil cell count (ANC) of about 2000 cells/μL or less.Thus, as used herein a subject suffering from neutropenia is one havingan ANC of about 2000 cells/μL or less, for example 1000 cells/μL or evenless than 500 cells/μL.

The term “non-insulin-dependent diabetes mellitus”; “NIDDM” or Type II”refer to diabetes that is characterized by hyperglycemia and insulinlevels being normal to high. Typically, Type II diabetes is a form ofdiabetes mellitus that has gradual onset in obese individuals over theage of 35. Insulin therapy is usually not required; however, Type IIdiabetics can be destined or prone to become fully insulin-dependent.

The term “oral administration” as used herein includes oral, buccal,enteral or intragastric administration.

The term “organ or tissue transplant rejection” as used herein refers toa consequence of organ or tissue transplantation caused by therecipient's or host's immune system in response to the transplantedorgan/tissue, which can damage or destroy it. Thus, one of skill in theart realizes that “organ or tissue transplant rejection” is controlledby the host subject.

The term “osteoporosis” as used herein is defined as a general term fordescribing any disease process that results in reduction in the mass ofbone.

The term “pain” as used herein refers to an unpleasant sensation. Forexample, the subject experiences discomfort, distress or suffering. Painof a moderate or high intensity is typically accompanied by anxiety.Thus, one of skill in the art is cognizant that pain may have dualproperties, for example sensation and emotion.

The term “parenteral administration” as used herein includes any form ofadministration in which the compound is absorbed into the subjectwithout involving absorption via the intestines. Exemplary parenteraladministrations that are used in the present invention include, but arenot limited to intramuscular, intravenous, intraperitoneal, intraocular,subcutaneous, or intraarticular or intratumoral administration. Yetfurther, parenteral administration also includes administration into asurgical field.

The term “peptide composition” as used herein refers to a compositioncomprising at least one of the lactoferrin related peptides of thepresent invention. Such peptides include, but are not limited to SEQ.ID. NO.1 (LFP-21), SEQ. ID. NO.2 (LFP-22), SEQ. ID. NO.3 (LFP-23), SEQ.ID. NO.4 (LFP-24), SEQ. ID. NO.5 (LFP-25), SEQ. ID. NO.6 (LFP-31), SEQ.ID. NO.7 (LFP-32), SEQ. ID. NO.8 (LFP-33), SEQ. ID. NO.9 (LFP-34), SEQ.ID. NO.10 (LFP-35), and SEQ. ID. NO.11 (LFP-36).

The term “pharmaceutically acceptable carrier” as used herein includesany and all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents and the like.The use of such media and agents for pharmaceutically active substancesis well known in the art. Except insofar as any conventional media oragent is incompatible with the vectors or cells of the presentinvention, its use in therapeutic compositions is contemplated.Supplementary active ingredients also can be incorporated into thecompositions.

The term “polynucleotide” is defined as a chain of nucleotides.Furthermore, nucleic acids are polymers of nucleotides. Thus, nucleicacids and polynucleotides as used herein are interchangeable. Oneskilled in the art has the general knowledge that nucleic acids arepolynucleotides, which can be hydrolyzed into the monomeric“nucleotides.” The monomeric nucleotides can be hydrolyzed intonucleosides. As used herein polynucleotides include, but are not limitedto, all nucleic acid sequences which are obtained by any means availablein the art, including, without limitation, recombinant means, i.e., thecloning of nucleic acid sequences from a recombinant library or a cellgenome, using ordinary cloning technology and PCR™, and the like, and bysynthetic means. Furthermore, one skilled in the art is cognizant thatpolynucleotides include mutations of the polynucleotides, include butare not limited to, mutation of the nucleotides, or nucleosides bymethods well known in the art.

The term “polypeptide” is defined as a chain of amino acid residues,usually having a defined sequence. As used herein the term polypeptideis interchangeable with the terms “peptides” and “proteins”.

The term “preventing” as used herein refers to minimizing, reducing orsuppressing the risk of developing a disease state or parametersrelating to the disease state or progression or other abnormal ordeleterious conditions.

The term “respiratory disorder” refers to any condition and/or disorderrelating to respiration and/or the respiratory system. The respiratorydisorder can be an allergic or non-allergic respiratory disorder. Morespecifically, the respiratory disorder includes, but is not limited toatopic asthma, non-atopic asthma, emphysema, bronchitis, chronicobstructive pulmonary disease, sinusitis and allergic rhinitis.

The term “sepsis” as used herein is defined as a Systemic InflammatoryResponse Syndrome to an infective process in which severe derangement ofthe host immune system fails to prevent extensive ‘spill over’ ofinflammatory mediators from a local infection focus into the systemiccirculation.

The term “septic shock” as used herein is a consequence of sepsis inwhich the systemic inflammatory response leads to the failure of vitalorgans' function (for example of the lungs as in ARDS).

The term “subject” as used herein, is taken to mean any mammaliansubject to which a lactoferrin composition is administered according tothe methods described herein. Thus, a skilled artisan realizes that amammalian subject, includes, but is not limited to humans, monkeys,horses, pigs, cows, dogs, cats, rats and mice. In a specific embodiment,the methods of the present invention are employed to treat a humansubject.

The term “Th₂ cells” as used herein is defined as a subset of CD4T-cells that are characterized by the cytokines they produce. Thesecells are mainly involved in stimulating B cells to produce antibody andare often called helper T-cells. It is also known that extracellularantigens tend to stimulate the production of Th₂ cells. Thus, as usedherein, “Th₂ cells” is interchangeable with “helper T-cells”.

The term “topical administration” as used herein includes, but is notlimited to topical, dermal, or epidermal.

The term “total cholesterol” as used herein refers to the sum of threekinds of lipids: high-density lipoprotein (HDL), low-density lipoprotein(LDL), and triglycerides. Levels of serum total cholesterol of >200mg/dl are levels that are an indicating risk factor for atherosclerosisand cardiovascular disease.

The term “treating” and “treatment” as used herein refers toadministering to a subject a therapeutically effective amount of arecombinant human lactoferrin composition so that the subject has animprovement in the said disease condition. The improvement is anyobservable or measurable improvement. Thus, one of skill in the artrealizes that a treatment may improve the patient condition, but may notbe a complete cure of the disease.

The term “triglycerides” as used herein are composed of fatty acidmolecules and are the basic chemicals contained in fats in both animalsand plants.

The term “type I osteoporosis” or “postmenopausal osteoporosis” as usedherein is usually found in women after the beginning of menopause. Theincidence in women is six to eight times higher than that in men. It hasbeen postulated that the cause of this osteoporosis is accelerated boneresorption. The increased bone turnover results in a secondary decreasein parathyroid hormone (PTH) secretion as well as a secondary reductionin the renal production of calcitriol. Patients present with trabecularbone loss with vertebral fractures or distal forearm fractures.

The term “type II osteoporosis” or “age-associated osteoporosis” or“senile osteoporosis” occurs in men or women over the age of 70. Themechanisms of this bone mass loss are thought to be increased PTHsecretion resulting from decreased gastrointestinal calcium absorptionand decreased osteoblast function. Patients usually present withfractures of the hip or vertebrae, sites that contain cortical andtrabecular bone, although fractures of the pelvis, ribs, and tibia canalso occur.

The term “very low density lipoprotein” or “VLDL” as used herein refersto a triglyceride carrying lipoprotein.

The term “wound” as used herein refers to any injury, such as an ulcer,as a result of disease or disorder, or as a result of an accident,incident, or surgical procedure. Wound can be further defined as acuteand/or chronic.

II. Lactoferrin

The lactoferrin can be obtained through isolation and purification fromnatural sources, for example, but not limited to mammalian milk. Thelactoferrin is preferably mammalian lactoferrin, such as bovine or humanlactoferrin. In preferred embodiments, the lactoferrin is producedrecombinantly using genetic engineering techniques well known and usedin the art, such as recombinant expression or direct production ingenetically altered animals, plants or eukaryotes, or chemicalsynthesis. See, e.g., U.S. Pat. Nos. 5,571,896; 5,571,697 and 5,571,691,which are herein incorporated by reference.

In certain aspects, the present invention provides lactoferrin variantshaving enhanced biological activities over natural LF and or rLF, e.g.,the ability to stimulate and/or inhibit cytokines or chemokines. Inparticular, the invention provides peptides related to lactoferrin,including deletional, substitutional or replacement variants.

A. Lactoferrin Related Peptides

In certain embodiments, the present invention concerns novel peptidecompositions. As used herein, a “peptide,” “amino acid molecule,”“polypeptide”, “peptide composition” generally refers, but is notlimited to, a peptide of about 33 amino acids. As used herein, all theterms related to “peptide” are interchangeable.

More particularly, the peptides of the present invention are derivedfrom lactoferrin. Thus, a lactoferrin related peptide includesrelated-compounds of the respective molecules that exhibit at least somebiological activity in common with their native counterparts, forexample lactoferrin. Such related-compounds include, but are not limitedto, truncated polypeptides and polypeptides having fewer amino acidsthan the native polypeptide. The full length lactoferrin protein, whichincludes polypeptide sequences, for example, but are not limited to SEQ.ID. NO.12 (GenBank accession AAA36159.1), SEQ. ID. NO.13 (GenBankaccession CAA38572.1); SEQ. ID. NO.14 (GenBank accessionNP_(—)032548.2); SEQ. ID. NO.15 (GenBank accession NP_(—)002334.1); SEQ.ID. NO.16 (GenBank accession NP_(—)851341.1); SEQ. ID. NO.17 (GenBankaccession AAN11304.1); SEQ. ID. NO.18 (GenBank accession AAN75578.2);SEQ. ID. NO.19 (GenBank accession CAA06441.1); SEQ. ID. NO.20 (GenBankaccession BAA13633.1); SEQ. ID. NO.21 (GenBank accession AAG48753.1);SEQ. ID. NO.22 (GenBank accession BAB03470.1); SEQ. ID. NO.23 (GenBankaccession AAF82241.1); SEQ. ID. NO.24 (GenBank accession CAB53387.1);SEQ. ID. NO.25 (GenBank accession BAA07458.1); SEQ. ID. NO.26 (GenBankaccession CAA09407.1); SEQ. ID. NO.27 (GenBank accession AAB60324.1);SEQ. ID. NO.28 (GenBank accession CAA55517.1); SEQ. ID. NO.29 (GenBankaccession CAA40366.1); SEQ. ID. NO.30 (GenBank accession CAA37914.1);SEQ. ID. NO.31 (GenBank accession CAA37116.1); SEQ. ID. NO.32 (GenBankaccession AAA97958.1); SEQ. ID. NO.33 (GenBank accession AAA59479.1);SEQ. ID. NO.34 (GenBank accession AAA59511.1); SEQ. ID. NO.35 (GenBankaccession AAA30610.1); SEQ. ID. NO.36 (GenBank accession AAA31102.1);SEQ. ID. NO.37 (GenBank accession AAA31059.1); SEQ. ID. NO.38 (GenBankaccession AAA30617.1); and SEQ. ID. NO.39 (GenBank accessionAAA30609.1), more fully described in U.S. Pat. Nos. 6,399,570;5,304,633; 5,317,084; 5,656,591; 5,849,885; 5,849,881; 5,766,939 whichare incorporated herein by reference in its entirety. More specifically,the lactoferrin related peptides may include the following sequences,but are not limited to these sequences, SEQ. ID. NO.1 (LFP-21), SEQ. ID.NO.2 (LFP-22), SEQ. ID. NO.3 (LFP-23), SEQ. ID. NO.4 (LFP-24), SEQ. ID.NO.5 (LFP-25), SEQ. ID. NO.6 (LFP-31), SEQ. ID. NO.7 (LFP-32), SEQ. ID.NO.8 (LFP-33), SEQ. ID. NO.9 (LFP-34), SEQ. ID. NO.10 (LFP-35), and SEQ.ID. NO.11 (LFP-36).

In certain embodiments the size of the peptide composition or amino acidmolecule may comprise, but is not limited to, about 1, about 2, about 3,about 4, about 5, about 6, about 7, about 8, about 9, about 10, about11, about 12, about 13, about 14, about 15, about 16, about 17, about18, about 19, about 20, about 21, about 22, about 23, about 24, about25, about 26, about 27, about 28, about 29, about 30, about 31, about32, about 33, about 34, about 35, about 36, about 37, about 38, about39, about 40, about 41, about 42, about 43, about 44, about 45, about46, about 47, about 48, about 49, about 50, about 60 or greater aminomolecule residues, and any range derivable therein.

As used herein, an “amino molecule” refers to any amino acid, amino acidderivative or amino acid mimic as would be known to one of ordinaryskill in the art. In certain embodiments, the residues of the peptideare sequential or contiguous, without any non-amino moleculeinterrupting the sequence of amino molecule residues. In otherembodiments, the sequence may comprise one or more non-amino moleculemoieties. In particular embodiments, the sequence of residues of thepeptide may be interrupted by one or more non-amino molecule moieties.

Accordingly, the term “peptide” encompasses amino molecule sequencescomprising at least one of the 20 common amino acids in naturallysynthesized proteins, or at least one modified or unusual amino acid,including but not limited to those shown on Table 1 below. TABLE 1Modified and Unusual Amino Acids Abbr. Amino Acid Abbr. Amino Acid Aad2-Aminoadipic acid EtAsn N-Ethylasparagine Baad 3-Aminoadipic acid HylHydroxylysine Bala β-alanine, β-Amino-propionic acid AHylallo-Hydroxylysine Abu 2-Aminobutyric acid 3Hyp 3-Hydroxyproline 4Abu4-Aminobutyric acid, 4Hyp 4-Hydroxyproline piperidinic acid Acp6-Aminocaproic acid Ide Isodesmosine Ahe 2-Aminoheptanoic acid AIleallo-Isoleucine Aib 2-Aminoisobutyric acid MeGly N-Methylglycine,sarcosine Baib 3-Aminoisobutyric acid MeIle N-Methylisoleucine Apm2-Aminopimelic acid MeLys 6-N-Methyllysine Dbu 2,4-Diaminobutyric acidMeVal N-Methylvaline Des Desmosine Nva Norvaline Dpm 2,2′-Diaminopimelicacid Nle Norleucine Dpr 2,3-Diaminopropionic acid Orn Ornithine EtGlyN-Ethylglycine

Peptide compositions may be made by any technique known to those ofskill in the art, including the expression of proteins, polypeptides orpeptides through standard molecular biological techniques, the isolationof peptide compounds from natural sources, or the chemical synthesis ofpeptides. The nucleotide and protein, polypeptide and peptide sequencesfor various genes have been previously disclosed, and may be found atcomputerized databases known to those of ordinary skill in the art. Onesuch database is the National Center for Biotechnology Information'sGenbank and GenPept databases. The coding regions for these known genesmay be amplified and/or expressed using the techniques disclosed hereinor as would be know to those of ordinary skill in the art.Alternatively, various commercial preparations of polypeptides andpeptides are known to those of skill in the art.

In certain embodiments a peptide composition may be purified. Generally,“purified” will refer to a specific polypeptide, or peptide compositionthat has been subjected to fractionation to remove various otherproteins, polypeptides, or peptides, and which composition substantiallyretains its activity, as may be assessed, for example, by the proteinassays, as would be known to one of ordinary skill in the art for thespecific or desired polypeptide or peptide.

B. Peptide Variants

Deletional variants can be produced by proteolysis of lactoferrin and/orexpression of a polynucleotide encoding a truncated lactoferrin asdescribed in U.S. Pat. No. 6,333,311, which is incorporated herein byreference.

Substitutional variants or replacement variants typically contain theexchange of one amino acid for another at one or more sites within theprotein. Substitutions can be conservative, that is, one amino acid isreplaced with one of similar shape and charge. Conservativesubstitutions are well known in the art and include, for example, thechanges of: alanine to serine; arginine to lysine; asparagine toglutamine or histidine; aspartate to glutamate; cysteine to serine;glutamine to asparagine; glutamate to aspartate; glycine to proline;histidine to asparagine or glutamine; isoleucine to leucine or valine;leucine to valine or isoleucine; lysine to arginine; methionine toleucine or isoleucine; phenylalanine to tyrosine, leucine or methionine;serine to threonine; threonine to serine; tryptophan to tyrosine;tyrosine to tryptophan or phenylalanine; and valine to isoleucine orleucine.

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte and Doolittle, 1982). It is accepted thatthe relative hydropathic character of the amino acid contributes to thesecondary structure of the resultant protein, which in turn defines theinteraction of the protein with other molecules, for example, enzymes,substrates, receptors, DNA, antibodies, antigens, and the like.

Each amino acid has been assigned a hydropathic index on the basis oftheir hydrophobicity and charge characteristics (Kyte and Doolittle,1982), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8);phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9);alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8);tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5);lysine (−3.9); and arginine (−4.5).

It is known in the art that certain amino acids may be substituted byother amino acids having a similar hydropathic index or score and stillresult in a protein with similar biological activity, e.g., still obtaina biological functionally equivalent protein. In making such changes,the substitution of amino acids whose hydropathic indices are within ±2is preferred, those that are within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101, incorporated herein by reference, states that thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with a biologicalproperty of the protein. As detailed in U.S. Pat. No. 4,554,101, thefollowing hydrophilicity values have been assigned to amino acidresidues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate(+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine(0); threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine(−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine(−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5);tryptophan (−3.4).

Still further, it is understood that an amino acid can be substitutedfor another having a similar hydrophilicity value and still obtains abiologically equivalent and immunologically equivalent protein. In suchchanges, the substitution of amino acids whose hydrophilicity values arewithin ±2 is preferred, those that are within ±1 are particularlypreferred, and those within ±0.5 are even more particularly preferred.

Thus, in the present invention, substitutional variants or replacementcan be produced using standard mutagenesis techniques, for example,site-directed mutagenesis as disclosed in U.S. Pat. Nos. 5,220,007;5,284,760; 5,354,670; 5,366,878; 5,389,514; 5,635,377; 5,789,166, and6,333,311, which are incorporated herein by reference. It is envisionedthat at least the N-terminal glycine amino acid residue can be replacedor substituted with any of the twenty natural occurring amino acids, forexample a positively charged amino acid (arginine, lysine, orhistidine), a neutral amino acid (alanine, asparagine, cysteine,glutamine, glycine, isoleucine, leucine, methionine, phenylaline,proline, serine, threonine, tryptophan, tyrosine, valine) and/or anegatively charged amino acid (aspartic acid or glutamic acid).

In terms of functional equivalents, it is well understood by the skilledartisan that, inherent in the definition of a “biologically functionalequivalent” protein is the concept that there is a limit to the numberof changes that may be made within a defined portion of the moleculewhile retaining a molecule with an acceptable level of equivalentbiological activity and/or enhancing the biological activity of thelactoferrin molecule. Biologically functional equivalents are thusdefined herein as those proteins in which selected amino acids (orcodons) may be substituted. Functional activity is defined as theability of the lactoferrin related peptide to stimulate or inhibitvarious cytokines or chemokines. In particular, a biological functionalequivalent will be able to stimulate production of macrophageinflammatory protein 3 alpha (MIP-3alpa).

Yet further, the lactoferrin peptides of this invention can be inglycosylated or unglycosylated form, can be modifiedpost-translationally (e.g., acetylation, and phosphorylation) or can bemodified synthetically (e.g., the attachment of a labeling group).Fragments of lactoferrin molecules that retain the prophylactic efficacycan also be used (see, e.g. PCT/IB00/00271, which is incorporated hereinin reference in its entirety).

C. Production of Lactoferrin Related Peptides

1. Synthetic Variants

The present invention also describes lactoferrin related peptides foruse in various embodiments of the present invention. Because of theirrelatively small size, the peptides of the invention can also besynthesized in solution or on a solid support in accordance withconventional techniques. Various automatic synthesizers are commerciallyavailable and can be used in accordance with known protocols. See, forexample, Stewart and Young (1984); Tam et al. (1983); Merrifield (1986);and Barany and Merrifield (1979), each incorporated herein by reference.Short peptide sequences, or libraries of overlapping peptides, usuallyfrom about 6 up to about 35 to 50 amino acids, which correspond to theselected regions described herein, can be readily synthesized and thenscreened in screening assays designed to identify reactive peptides.Alternatively, recombinant DNA technology may be employed wherein anucleotide sequence which encodes a peptide of the invention is insertedinto an expression vector, transformed or transfected into anappropriate host cell and cultivated under conditions suitable forexpression.

2. Recombinant Synthesis

a) Nucleic Acid Compositions

Certain embodiments of the present invention concern a nucleic acidcomposition having a nucleic acid sequence of lactoferrin. In certainaspects, a lactoferrin nucleic acid comprises a wild-type or a mutantlactoferrin nucleic acid. In particular aspects, a lactoferrin nucleicacid sequence encodes for or comprises a transcribed nucleic acid. Inother aspects, a lactoferrin nucleic acid sequence comprises a nucleicacid segment of SEQ. ID. NO.40 (GenBank accession M93150); SEQ. ID.NO.41 (GenBank accession X54801); SEQ. ID. NO.42 (GenBank accessionNM_(—)008522); SEQ. ID. NO.43 (GenBank accession NM_(—)002343); SEQ. ID.NO.44 (GenBank accession NM_(—)180998); SEQ. ID. NO.45 (GenBankaccession AY137470); SEQ. ID. NO.46 (GenBank accession AY178998); SEQ.ID. NO.47 (GenBank accession AJ005203); SEQ. ID. NO.48 (GenBankaccession D88510); SEQ. ID. NO.49 (GenBank accession AF332168); SEQ. ID.NO.50 (GenBank accession AB046664); SEQ. ID. NO.51 (GenBank accessionAAF82241.1); SEQ. ID. NO.52 (GenBank accession AJ131674); SEQ. ID. NO.53(GenBank accession D38380); SEQ. ID. NO.54 (GenBank accession AJ010930);SEQ. ID. NO.55 (GenBank accession U07643); SEQ. ID. NO.56 (GenBankaccession X78902); SEQ. ID. NO.57 (GenBank accession X57084); SEQ. ID.NO.58 (GenBank accession X53961); SEQ. ID. NO.59 (GenBank accessionX52941); SEQ. ID. NO.60 (GenBank accession U53857); SEQ. ID. NO.61(GenBank accession M73700); SEQ. ID. NO.62 (GenBank accession M83202);SEQ. ID. NO.63 (GenBank accession L19981); SEQ. ID. NO.64 (GenBankaccession M92089); SEQ. ID. NO.65 (GenBank accession M81327); SEQ. ID.NO.66 (GenBank accession M63502); and SEQ. ID. NO.67 (GenBank accessionL08604) or a biologically functional equivalent thereof. In particularaspects, the nucleic acid sequence of the present invention encodes apolypeptide, peptide, for example, but not limited to SEQ. ID. NO.1(LFP-21), SEQ. ID. NO.2 (LFP-22), SEQ. ID. NO.3 (LFP-23), SEQ. ID. NO.4(LFP-24), SEQ. ID. NO.5 (LFP-25), SEQ. ID. NO.6 (LFP-31), SEQ. ID. NO.7(LFP-32), SEQ. ID. NO.8 (LFP-33), SEQ. ID. NO.9 (LFP-34), SEQ. ID. NO.10(LFP-35), and SEQ. ID. NO.11 (LFP-36). Thus, nucleic acid compositionsencoding lactoferrin and/or lactoferrin related peptides are hereinprovided and are also available to a skilled artisan at accessibledatabases, including the National Center for Biotechnology Information'sGenBank database and/or commercially available databases, such as fromCelera Genomics, Inc. (Rockville, Md.).

The term “nucleic acid” is well known in the art. A “nucleic acid” asused herein will generally refer to a molecule (i.e., a strand) of DNA,RNA or a derivative or analog thereof, comprising a nucleobase. Anucleobase includes, for example, a naturally occurring purine orpyrimidine base found in DNA (e.g., an adenine “A,” a guanine “G,” athymine “T” or a cytosine “C”) or RNA (e.g., an A, a G, an uracil “U” ora C). The term “nucleic acid” encompass the terms “oligonucleotide” and“polynucleotide,” each as a subgenus of the term “nucleic acid.” Theterm “oligonucleotide” refers to a molecule of between about 3 and about100 nucleobases in length. The term “polynucleotide” refers to at leastone molecule of greater than about 100 nucleobases in length.

These definitions generally refer to a single-stranded molecule, but inspecific embodiments will also encompass an additional strand that ispartially, substantially or fully complementary to the single-strandedmolecule. Thus, a nucleic acid may encompass a double-stranded moleculeor a triple-stranded molecule that comprises one or more complementarystrand(s) or “complement(s)” of a particular sequence comprising amolecule. As used herein, a single stranded nucleic acid may be denotedby the prefix “ss,” a double stranded nucleic acid by the prefix “ds,”and a triple stranded nucleic acid by the prefix “ts.”

The nucleic acids of the present invention encompass biologicallyfunctional equivalent lactoferrin proteins, polypeptides, or lactoferrinrelated peptides. Examples of such peptides include, but are not limitedto SEQ. ID. NO.1 (LFP-21), SEQ. ID. NO.2 (LFP-22), SEQ. ID. NO.3(LFP-23), SEQ. ID. NO.4 (LFP-24), SEQ. ID. NO.5 (LFP-25), SEQ. ID. NO.6(LFP-31), SEQ. ID. NO.7 (LFP-32), SEQ. ID. NO.8 (LFP-33), SEQ. ID. NO.9(LFP-34), SEQ. ID. NO.10 (LFP-35), and SEQ. ID. NO.11 (LFP-36). Suchsequences may arise as a consequence of codon redundancy or functionalequivalency that is known to occur naturally within nucleic acidsequences or the proteins, polypeptides or peptides thus encoded.Alternatively, functionally equivalent proteins, polypeptides orpeptides may be created via the application of recombinant DNAtechnology, in which changes in the protein, polypeptide or peptidestructure may be engineered, based on considerations of the propertiesof the amino acids being exchanged. Changes designed by man may beintroduced, for example, through the application of site-directedmutagenesis techniques as discussed herein below, e.g., to introduceimprovements or alterations to the antigenicity of the protein,polypeptide or peptide.

Encompassed by the invention are nucleic acid sequences encodingrelatively small peptides, such as, for example, peptides of from about3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about11, about 12, about 13, about 14, about 15, about 16, about 17, about18, about 19, about 20, about 21, about 22, about 23, about 24, about25, about 26, about 27, about 28, about 29, about 30, about 31, about32, about 33, about 34, about 35, about 35, about 36, about 37, about38, about 39, about 40, about 41, about 42, about 43, about 44, about45, about 46, about 47, about 48, about 49, to about 50 amino acids inlength, or more preferably, of from about 1 to about 33 amino acids inlength; as set forth in SEQ. ID. NO. 1, SEQ. ID. NO.2, SEQ. ID. NO.4,SEQ. ID. NO.5, SEQ. ID. NO.6, SEQ. ID. NO.7, SEQ. ID. NO.8, SEQ. ID.NO.9, SEQ. ID. NO.10, and SEQ. ID. NO.11 and also larger peptides up toand from about 1 to about 34 amino acids in length; as set forth in SEQ.ID. NO.3.

3. Expression Vectors

The present invention may involve using expression constructs as thepharmaceutical composition. In certain embodiments, it is contemplatedthat the expression construct comprises polynucleotide sequencesencoding polypeptides which can enhance the immune system, for examplethe expression construct may comprise a nucleic acid sequence encodingthe amino acid sequence of SEQ. ID. NO.1 (LFP-21), SEQ. ID. NO.2(LFP-22), SEQ. ID. NO.3 (LFP-23), SEQ. ID. NO.4 (LFP-24), SEQ. ID. NO.5(LFP-25), SEQ. ID. NO.6 (LFP-31), SEQ. ID. NO.7 (LFP-32), SEQ. ID. NO.8(LFP-33), SEQ. ID. NO.9 (LFP-34), SEQ. ID. NO.10 (LFP-35), SEQ. ID.NO.11 (LFP-36), SEQ. ID. NO.12.

In certain embodiments, the present invention involves the manipulationof genetic material to produce expression constructs that encode thepeptides of the present invention. Thus, the peptides are contained inan expression vector. Such methods involve the generation of expressionconstructs containing, for example, a heterologous nucleic acid sequenceencoding the peptide of interest and a means for its expression,replicating the vector in an appropriate cell, obtaining viral particlesproduced therefrom, and infecting cells with the recombinant virusparticles.

As used in the present invention, the term “expression vector” refers toany type of genetic construct comprising a nucleic acid coding for alactoferrin related peptide, for example, but not limited to SEQ. ID.NO.1, SEQ. ID. NO.2, SEQ. ID. NO.3, SEQ. ID. NO.4, SEQ. ID. NO.5, SEQ.ID. NO.6, SEQ. ID. NO.7, SEQ. ID. NO.8, SEQ. ID. NO.9, SEQ. ID. NO.10,and SEQ. ID. NO.11. In some cases, DNA molecules are then translatedinto a protein, polypeptide, or peptide. Expression vectors can containa variety of “control sequences,” which refer to nucleic acid sequencesnecessary for the transcription and possibly translation of an operablylinked coding sequence in a particular host cell. In addition to controlsequences that govern transcription and translation, vectors andexpression vectors may contain nucleic acid sequences that serve otherfunctions as well and are described infra. It is contemplated in thepresent invention, that virtually any type of vector may be employed inany known or later discovered method to deliver nucleic acids encoding alactoferrin related peptide. An expression vector comprising a nucleicacid encoding a lactoferrin related peptide may comprise a virus orengineered construct derived from a viral genome.

In particular embodiments of the invention, a plasmid vector iscontemplated for use to transform a host cell. In general, plasmidvectors containing replicon and control sequences that are derived fromspecies compatible with the host cell are used in connection with thesehosts. The vector ordinarily carries a replication site, as well asmarking sequences that are capable of providing phenotypic selection intransformed cells. Plasmid vectors are well known and are commerciallyavailable. Such vectors include, but are not limited to, thecommercially available pSupervector (OligoEngine, Seattle, Wash.), andpSuppressor Neo vector (IMGENEX Corporation). Other vectors that may beemployed in the present invention include, but are not limited to, thefollowing eukaryotic vectors: pWLNEO, pSV2CAT, pOG44, PXT1, pSG(Stratagene) pSVK3, pBSK, pBR322, pUC vectors, vectors that containmarkers that can be selected in mammalian cells, such as pcDNA3.1,episomally replicating vectors, such as the pREP series of vectors,pBPV, pMSG, pSVL (Pharmacia), adenovirus vector (AAV; pCWRSV, Chatterjeeet al. (1992)); retroviral vectors, such as the pBABE vector series, aretroviral vector derived from MoMuLV (pGlNa, Zhou et al., (1994)); andpTZ18U (BioRad, Hercules, Calif.).

In one embodiment, a gene encoding a lactoferrin related peptide isintroduced in vivo in a viral vector. The ability of certain viruses toenter cells via receptor-mediated endocytosis and to integrate into thehost cell genome and express viral genes stably and efficiently havemade them attractive candidates for the transfer of foreign genes intomammalian cells (Ridgeway, 1988; Nicolas and Rubenstein, 1988; Baichwaland Sugden, 1986; Temin, 1986). Such vectors include an attenuated ordefective DNA virus, such as but not limited to herpes simplex virus(HSV), papilloma virus, Epstein Barr virus (EBV), adenovirus,adeno-associated virus (AAV), lentivirus and the like. Defectiveviruses, which entirely or almost entirely lack viral genes, arepreferred. Defective virus is not infective after introduction into acell. Use of defective viral vectors allows for administration to cellsin a specific, localized area, without concern that the vector caninfect other cells. Thus, any tissue can be specifically targeted.Examples of particular vectors include, but are not limited to, adefective herpes virus 1 (HSV1) vector (Kaplitt et al., 1991) anattenuated adenovirus vector, (Stratford-Perricaudet et al., 1992), adefective adeno-associated virus vector (Samulski et al., 1987 andSamulski et al., 1989) or pox-vector. Such vectors may be used to (i)transform cell lines in vitro for the purpose of expressing the alactoferrin related peptide or (ii) to transform cells in vitro or invivo to provide therapeutic molecules for gene therapy. Thus, thepresent invention contemplates viral vectors such as, but not limitedto, an adenoviral vector, an adeno-associated viral vector, a retroviralvector, a lentiviral vector, a herpes viral vector, polyoma viralvector, pox-vector or hepatitis B viral vector.

Preferably, for in vivo administration, an appropriate immunosuppressivetreatment is employed in conjunction with the viral vector, e.g.,adenovirus vector, to avoid immunodeactivation of the viral vector andtransfected cells. For example, immunosuppressive cytokines, such asinterleukin-12 (IL-12), interferon-γ (IFN-γ), or anti-CD4 antibody, canbe administered to block humoral or cellular immune responses to theviral vectors (Wilson, Nature Medicine (1995). In addition, it isadvantageous to employ a viral vector that is engineered to express aminimal number of antigens.

In another embodiment the gene can be introduced in a retroviral vector,e.g., as described in U.S. Pat. No. 5,399,346; Mann et al., 1983; U.S.Pat. No. 4,650,764; U.S. Pat. No. 4,980,289; Markowitz et al., 1988;U.S. Pat. No. 5,124,263; International Patent Publication No. WO95/07358; and Kuo et al., 1993, each of which is incorporated herein byreference in its entirety. Targeted gene delivery is described inInternational Patent Publication WO 95/28494.

Alternatively, the vector can be introduced in vivo by lipofection. Forthe past decade, there has been increasing use of liposomes forencapsulation and transfection of nucleic acids in vitro. Syntheticcationic lipids designed to limit the difficulties and dangersencountered with liposome mediated transfection can be used to prepareliposomes for in vivo transfection of a gene encoding a marker. The useof cationic lipids may promote encapsulation of negatively chargednucleic acids, and also promote fusion with negatively charged cellmembranes. The use of lipofection to introduce exogenous genes into thespecific organs in vivo has certain practical advantages. Moleculartargeting of liposomes to specific cells represents one area of benefit.Lipids may be chemically coupled to other molecules for the purpose oftargeting. Targeted peptides, e.g., hormones or neurotransmitters, andproteins such as antibodies, or non-peptide molecules could be coupledto liposomes chemically.

It is also possible to introduce the vector in vivo as a naked DNAplasmid. Naked DNA vectors for gene therapy can be introduced into thedesired host cells by methods known in the art, e.g., transfection,electroporation, microinjection, transduction, cell fusion, DEAEdextran, calcium phosphate precipitation, use of a gene gun, or use of aDNA vector transporter (Wu and Wu, 1988).

As used herein, the terms “cell,” “cell line,” and “cell culture” may beused interchangeably. All of these terms also include their progeny,which is any and all subsequent generation formed by cell division. Itis understood that all progeny may not be identical due to deliberate orinadvertent mutations. A host cell may be “transfected” or“transformed,” which refers to a process by which exogenous nucleic acidis transferred or introduced into the host cell. A transformed cellincludes the primary subject cell and its progeny. As used herein, theterms “engineered” and “recombinant” cells or host cells are intended torefer to a cell into which an exogenous nucleic acid sequence, such as,for example, a lactoferrin related peptide. Therefore, recombinant cellsare distinguishable from naturally occurring cells that do not contain arecombinantly introduced nucleic acid.

Host cells may be derived from prokaryotes or eukaryotes, depending uponwhether the desired result is replication of the vector or expression ofpart or all of the vector encoded nucleic acid sequences. Numerous celllines and cultures are available for use as a host cell, and they can beobtained through the American Type Culture Collection (ATCC), which isan organization that serves as an archive for living cultures andgenetic materials. In certain embodiments, a cell may comprise, but isnot limited to, at least one skin, bone, neuron, axon, cartilage, bloodvessel, cornea, muscle, facia, brain, prostate, breast, endometrium,lung, pancreas, small intestine, blood, liver, testes, ovaries, cervix,colon, skin, stomach, esophagus, spleen, lymph node, bone marrow,kidney, peripheral blood, embryonic or ascite cell, and all cancersthereof. An appropriate host can be determined by one of skill in theart based on the vector backbone and the desired result. Bacterial cellsused as host cells for vector replication and/or expression includeDH5α, JM109, and KC8, as well as a number of commercially availablebacterial hosts such as SURE® Competent Cells and SOLOPACK™ Gold Cells(STRATAGENE®, La Jolla).

Examples of eukaryotic host cells for replication and/or expression of avector include HeLa, NIH3T3, Jurkat, 293, Cos, CHO, Saos, and PC12.Other eukaryotic cells include yeast cells, such as Aspergillus speciesand Saccharomyces species. Many host cells from various cell types andorganisms are available and would be known to one of skill in the art.In addition, a host cell strain may be chosen that modulates theexpression of the inserted sequences, and/or modifies and/or processesthe gene product in the specific fashion desired. Such modifications(e.g., glycosylation) and/or processing (e.g., cleavage) of proteinproducts may be important for the function of the protein.

III. Pharmaceutical Compositions

The present invention is drawn to a composition comprising a lactoferrinthat is dispersed in a pharmaceutical carrier. The lactoferrin that iscontained in the composition of the present invention compriseslactoferrin related peptides, for example, SEQ. ID. NO. 1, SEQ. ID.NO.2, SEQ. ID. NO.3, SEQ. ID. NO.4, SEQ. ID. NO.5, SEQ. ID. NO.6, SEQ.ID. NO.7, SEQ. ID. NO.8, SEQ. ID. NO.9, SEQ. ID. NO.10, and SEQ. ID.NO.11 and/or expression vectors encoding a lactoferrin related peptide.

Yet further, the composition comprises lactoferrin related peptides incombination with a metal chelator dispersed in a pharmaceutical carrier.Thus, the present invention is drawn to a composition with or without ametal chelator that is dispersed in a pharmaceutical carrier. One ofskill in the art understands that both compositions (e.g., lactoferrinrelated peptides alone or in combination with a metal chelator) arewithin the scope of the present invention and can be usedinterchangeably depending upon the type of response that is desired. Itis envisioned that the addition of a metal chelator to the compositionenhances the sequestering of metal ions and thus strengthens the immunesystem or enhances the effect of the lactoferrin related peptides.

Metal chelators that can be used in combination with lactoferrin,include the divalent metal chelators, for example,ethylenediaminetetraacetic acid (EDTA),[ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA),1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA),hydroxyethlene triamine diacetic acid, (HEDTA) or any salts thereof. Theamount of the metal chelator in the composition may vary from about 0.01μg to about 20 g. More preferably, EDTA is used in combination withlactoferrin.

The lactoferrin composition of the present invention may comprisedifferent types of carriers depending on whether it is to beadministered in solid, liquid or aerosol form, and whether it need to besterile for such routes of administration as injection. The presentinvention can be administered intravenously, intradermally,transdermally, intrathecally, intraarterially, intraperitoneally,intranasally, intravaginally, intrarectally, topically, intramuscularly,subcutaneously, mucosally, orally, topically, locally, inhalation (e.g.,aerosol inhalation), injection, infusion, continuous infusion, localizedperfusion bathing target cells directly, via a catheter, via a lavage,in cremes, in lipid compositions (e.g., liposomes), or by other methodor any combination of the forgoing as would be known to one of ordinaryskill in the art (see, for example, Remington's Pharmaceutical Sciences,18th Ed. Mack Printing Company, 1990, incorporated herein by reference).

The composition of the present invention suitable for administration isprovided in a pharmaceutically acceptable carrier with or without aninert diluent. The carrier should be assimilable and includes liquid,semi-solid, e.g., pastes, or solid carriers. Except insofar as anyconventional media, agent, diluent or carrier is detrimental to therecipient or to the therapeutic effectiveness of the compositioncontained therein, its use in administrable composition for use inpracticing the methods of the present invention is appropriate. Examplesof carriers or diluents include fats, oils, water, saline solutions,lipids, liposomes, resins, binders, fillers and the like, orcombinations thereof.

Sterile injectable solutions are prepared by incorporating thelactoferrin in the required amount in the appropriate solvent withvarious of the other ingredients enumerated above, as required, followedby filtered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

In a specific embodiment of the present invention, the composition iscombined or mixed thoroughly with a semi-solid or solid carrier. Themixing can be carried out in any convenient manner such as grinding.Stabilizing agents can be also added in the mixing process in order toprotect the composition from loss of therapeutic activity, i.e.,denaturation in the stomach or in the open wound environment. Examplesof stabilizers for use in an the composition include buffers, aminoacids such as glycine and lysine, carbohydrates such as dextrose,mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol,mannitol, etc., proteolytic enzyme inhibitors, and the like. Yetfurther, it is envisioned that divalent metal chelators, for exampleEDTA, can also be used to stabilize the composition of the presentinvention. More preferably, for an orally administered composition, thestabilizer can also include antagonists to the secretion of stomachacids. Yet further, for a topically administered composition, thestabilizer can also include antagonists to skin acids.

In further embodiments, the present invention may concern the use of apharmaceutical lipid vehicle compositions that include lactoferrin orlactoferrin related peptides, one or more lipids, and an aqueoussolvent. As used herein, the term “lipid” will be defined to include anyof a broad range of substances that is characteristically insoluble inwater and extractable with an organic solvent. This broad class ofcompounds are well known to those of skill in the art, and as the term“lipid” is used herein, it is not limited to any particular structure.Examples include compounds which contain long-chain aliphatichydrocarbons and their derivatives. A lipid may be naturally occurringor synthetic (i.e., designed or produced by man). However, a lipid isusually a biological substance. Biological lipids are well known in theart, and include for example, neutral fats, phospholipids,phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids,glycolipids, sulphatides, lipids with ether and ester-linked fatty acidsand polymerizable lipids, and combinations thereof. Of course, compoundsother than those specifically described herein that are understood byone of skill in the art as lipids are also encompassed by thecompositions and methods of the present invention.

One of ordinary skill in the art would be familiar with the range oftechniques that can be employed for dispersing a composition in a lipidvehicle. For example, the lactoferrin or lactoferrin related peptidesmay be dispersed in a solution containing a lipid, dissolved with alipid, emulsified with a lipid, mixed with a lipid, combined with alipid, covalently bonded to a lipid, contained as a suspension in alipid, contained or complexed with a micelle or liposome, or otherwiseassociated with a lipid or lipid structure by any means known to thoseof ordinary skill in the art. The dispersion may or may not result inthe formation of liposomes.

The composition for oral administration which is combined with asemi-solid or solid carrier can be further formulated into hard or softshell gelatin capsules, tablets, or pills. More preferably, gelatincapsules, tablets, or pills are enterically coated. Enteric coatingsprevent denaturation of the composition in the stomach or upper bowelwhere the pH is acidic. See, e.g., U.S. Pat. No. 5,629,001. Uponreaching the small intestines, the basic pH therein dissolves thecoating and permits the lactoferrin composition to be released andabsorbed by specialized cells, e.g., epithelial enterocytes and Peyer'spatch M cells.

Oral administration of the composition includes oral, buccal, enteral orintragastric administration. It is also envisioned that the compositionmay be used as a food additive. For example, the composition issprinkled on food or added to a liquid prior to ingestion. In furtherembodiments, the oral composition is administered in conjunction with anantacid. Thus, an antacid is administered prior or substantiallysimultaneously with or after oral administration of the composition. Theadministration of an antacid just prior or immediately following theadministration of the composition may help to reduce the degree ofinactivation of the lactoferrin in the digestive tract. Examples ofappropriate antacids include, but are not limited to, sodiumbicarbonate, magnesium oxide, magnesium hydroxide, calcium carbonate,magnesium trisilicate, magnesium carbonate and alumin hydroxide gel.

In another embodiment, a powdered composition is combined with a liquidcarrier such as, i.e., water or a saline solution, with or without astabilizing agent.

In other preferred embodiments of the invention, lactoferrin orlactoferrin related peptides may be formulated for administration viavarious miscellaneous routes, for example, topical (i.e., transdermal)administration, mucosal administration (intranasal, vaginal, etc.)and/or inhalation.

Pharmaceutical compositions for topical administration may include theactive compound formulated for a medicated application such as anointment, paste, cream or powder. Ointments include all oleaginous,adsorption, emulsion and water-solubly based compositions for topicalapplication, while creams and lotions are those compositions thatinclude an emulsion base only. Topically administered medications maycontain a penetration enhancer to facilitate adsorption of the activeingredients through the skin. Suitable penetration enhancers includeglycerin, alcohols, alkyl methyl sulfoxides, pyrrolidones andlaurocapram. Possible bases for compositions for topical applicationinclude polyethylene glycol, lanolin, cold cream and petrolatum as wellas any other suitable absorption, emulsion or water-soluble ointmentbase. Topical preparations may also include emulsifiers, gelling agents,and antimicrobial preservatives as necessary to preserve the activeingredient and provide for a homogenous mixture. Transdermaladministration of the present invention may also comprise the use of a“patch”. For example, the patch may supply one or more active substancesat a predetermined rate and in a continuous manner over a fixed periodof time.

In certain embodiments, the pharmaceutical compositions may be deliveredby eye drops, intranasal sprays, inhalation, and/or other aerosoldelivery vehicles. Methods for delivering compositions directly to thelungs via nasal aerosol sprays have been described e.g., in U.S. Pat.Nos. 5,756,353 and 5,804,212 (each specifically incorporated herein byreference in its entirety). Likewise, the delivery of drugs usingintranasal microparticle resins (Takenaga et al., 1998) andlysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725,871,specifically incorporated herein by reference in its entirety) are alsowell-known in the pharmaceutical arts. Likewise, transmucosal drugdelivery in the form of a polytetrafluoroethylene support matrix isdescribed in U.S. Pat. No. 5,780,045 (specifically incorporated hereinby reference in its entirety).

The term aerosol refers to a colloidal system of finely divided solid ofliquid particles dispersed in a liquefied or pressurized gas propellant.The typical aerosol of the present invention for inhalation will consistof a suspension of active ingredients in liquid propellant or a mixtureof liquid propellant and a suitable solvent. Suitable propellantsinclude hydrocarbons and hydrocarbon ethers. Suitable containers willvary according to the pressure requirements of the propellant.Administration of the aerosol will vary according to subject's age,weight and the severity and response of the symptoms.

In further embodiments, the compositions of this invention, can beincorporated into various types of ophthalmic formulations for deliveryto the eye (e.g., topically, intracamerally, or via an implant). Thecompositions are preferably incorporated into topical ophthalmicformulations for delivery to the eye. The compositions may be combinedwith ophthalmologically acceptable preservatives, surfactants, viscosityenhancers, penetration enhancers, buffers, sodium chloride, and water toform an aqueous, sterile ophthalmic suspension or solution. Ophthalmicsolution formulations may be prepared by dissolving a compositions in aphysiologically acceptable isotonic aqueous buffer. Further, theophthalmic solution may include an ophthalmologically acceptablesurfactant to assist in dissolving the compositions. Furthermore, theophthalmic solution may contain an agent to increase viscosity, such as,hydroxymethylcellulose, hydroxyethylcellulose,hydroxypropylmethylcellulose, methylcellulose, polyvinylpyrrolidone, orthe like, to improve the retention of the formulation in theconjunctival sac. Gelling agents can also be used, including, but notlimited to, gellan and xanthan gum. In order to prepare sterileophthalmic ointment formulations, the active ingredient is combined witha preservative in an appropriate vehicle, such as, mineral oil, liquidlanolin, or white petrolatum. Sterile ophthalmic gel formulations may beprepared by suspending the compositions in a hydrophilic base preparedfrom the combination of, for example, carbopol-974, or the like,according to the published formulations for analogous ophthalmicpreparations; preservatives and tonicity agents can be incorporated.

Further, the composition for topical administration which is combinedwith a semi-solid carrier can be further formulated into a gel ointment.A preferred carrier for the formation of a gel ointment is a gelpolymer. Gel polymers prevent denaturation of the composition in theopen skin by serum proteases. The gel formulation of the presentinvention also provides a controlled delivery system for lactoferrin orits activity on a wound site. Controlled delivery refers to drug releaseor activity release sufficient to maintain a therapeutic level over anextended period of time, such as up to 24 hours or more, preferably inthe range of 1 to 12 hours. The present gel formulation increases thecontact time of the lactoferrin at the wound site and provides asustained release dosage form necessary to achieve a significantincrease in the rate of wound healing. This is an important advantagebecause it permits less frequent application of the formulation to thewound and thereby permits fewer disturbances to the wound and itscellular components.

The gel formulation of the present invention has the advantage ofadhering to a wound and conforming to irregular body or wound contours.The gels may be applied directly to a wound site or in conjunction witha compliant porous or microporous substrate, for example in the form ofa coating, to be applied to the wound site. Gels have the furtheradvantages of having a high water content (which keeps the wound moist),the ability to absorb wound exudate, easy application to a wound andeasy removal by washing. Gels have a cool feeling when applied to awound and thus can increase patient comfort and acceptance of theformulation, especially on sensitive wounds.

The aqueous gels of the present invention have different viscositiesdepending on the intended application of the gel. Viscosity is a measureof the resistance of a liquid to flow. It is defined as the ratio of theshearing stress to the rate of shearing. The shear stress is theresistance of the liquid to flow under the influence of an appliedforce, i.e., the molecular resistance within a body opposing an externalforce. The shear stress is defined as the ratio of the force to the areasheared. When a liquid is sheared, assuming laminar flow, the layers ofthe liquid move at different rates. The relative rate of motion of thelayers is only one factor in the rate of shear. The other is thedistance, or clearance between the shearing planes. Thus, shear rate isdefined as the ratio of the velocity of the gel to the clearance.Viscosity has the dimensions of dynes/sec/cm². These dimensions arereferred to as poise. The dimensions of viscosity referred to herein,unless otherwise indicated, are in centipoise (cP) as measured using aBrookfield viscometer. All viscosity values are at room temperature,e.g., 22° C.-25° C., unless otherwise indicated.

The gel forming materials of the present invention may be water-solublepolymers capable of forming a viscous aqueous solution or non-watersoluble, water swellable polymers (e.g., collagen), which can also forma viscous solution. Swellable polymers are those that absorb waterrather than dissolve in water. Cross-linked forms of the polymerdescribed herein may not be water soluble but may be water-swellable.Therefore, cross-linked forms of the polymer are within the scope of thepresent invention. Cross-linking refers to covalently bonding polymerchains together with a bifunctional reagent such as glutaraldehyde.Also, it is understood by those skilled in the art that certain polymersmay have to be used in the salt form or partially neutralized in orderto be made water soluble. For example, it is preferable to usehyaluronic acid as sodium hyaluronate to provide suitable watersolubility.

In the aqueous gel formulations for topical or incisional wound healing,the polymer may be selected from the group consisting of vinyl polymers,polyoxyethylene-polyoxypropylene copolymers, polysaccharides, proteins,poly(ethylene oxide), acrylamide polymers and derivatives or saltsthereof. It is understood that poly(ethyleneoxide) includes polyethyleneglycol. In the gel formulations for use in healing wounds in theanterior chamber of the eye, the polymers may be the same except that itis not preferred to use the polyoxyethylene-polyoxypropylene copolymersor poly(ethylene oxide). Also, for anterior chamber use, it is preferredthat the polymer is biodegradable, i.e., it will break down intoharmless constituents that can be drained from or metabolized in theanterior chamber. In the low viscosity, aqueous formulations for use inophthalmic wound healing, the gel forming polymers may be the same asfor topical or incisional wound healing, except that poly(ethyleneoxide) is not preferred to be used.

The vinyl polymers useful in the present invention may be selected fromthe group consisting of polyacrylic acid, polymethacrylic acid,polyvinyl pyrrolidone and polyvinyl alcohol. The polysaccharides usefulin the present invention are selected from the group consisting ofcellulose or cellulose derivatives, glycosaminoglycans, agar, pectin,alginic acid, dextran, starch, and chitosan. Starch occurs in two forms,α-amylose and amylopectin. The more water-soluble α-amylose ispreferred. The glycosaminoglycans are selected from the group consistingof hyaluronic acid, chondroitin, chondroitin-4-sulfate,chondroitin-6-sulfate, dermatan sulfate, keratan sulfate, heparinsulfate and heparin. The glycosaminoglycans are used to enhance woundhealing in combination with any other gel-forming polymer. The proteinsuseful in the present invention are selected from the group consistingof collagen, gelatin and fibronectin. The acrylamide polymers arepolyacrylamide or polymethacrylamide polymers. Biocompatiblepolyacrylamide polymers are preferred. In further embodiments, carbomersare the preferred polyacrylamide polymer. Carbomers are synthetic highmolecular weight polymers of acrylic acid cross linked with either alkylesters of sucrose or pentaerythritol. Suitable commercially availablegrades of carbomer include Carbopol 910, Carbopol 934P, Carbopol 940,Carbopol 941, Carbopol 971P, Carbopol 974P, Carbopol 980, Carbopol 981,Carbopol 1342, Rheogic 252L, Rheogic 250H, and Hostacerin PN73.

In the gel formulation for topical or incisional wound healing, theviscosity may be within the range 1,000-12,000,000 cps at roomtemperature. It is preferred that the viscosity range be50,000-2,000,000. In one embodiment of the present invention, thetopical gel formulation may comprise 0.01-5% by weight polyacrylic acidhaving an average molecular weight of about 450,000-4,000,000. In apreferred embodiment, the polyacrylic acid is present at 0.5-1.5% byweight and has an average molecular weight of 2,000,000-4,000,000. ThepH of the polyacrylic acid gel should be within the range 4.5-8 and morepreferably in the range 6.5-7.5.

In another embodiment, the topical and incisional gel of the presentinvention may comprise 15-60% by weight of apolyoxyethylene-polyoxypropylene block copolymer having an averagemolecular weight of about 500-50,000. In a preferred embodiment, theblock copolymer is present at 15-40% by weight and has an averagemolecular weight in the range 1,000-15,000. The block copolymers used inthe present invention are commonly known as Pluronics. PreferredPluronics are Pluronic F88 and F127.

In a further embodiment, the topical or incisional gel may comprise 1 to20% by weight of a cellulose polymer having a molecular weight of about50,000 to 700,000. In a preferred embodiment, the cellulose polymer ispresent at 2-8% by weight and has an average molecular weight in therange 80,000-240,000. Preferred cellulose polymers arehydroxypropylmethyl cellulose (HPMC), carboxymethyl cellulose (CMC) andmethyl cellulose (MC).

In a further embodiment, the topical and incisional gel may comprise0.5-10% by weight of hyaluronic acid having an average molecular weightin the range 500,000 to 8,000,000. In a preferred embodiment, thehyaluronic acid is present at 1.5-6.0% by weight and the averagemolecular weight is greater than 1,000,000.

Acrylamide polymers may be useful for all types of wound healing,particularly in the anterior chamber of the eye. An absorbableacrylamide polymer, such as polyacrylamide, may be a good substitute forpresent carrier systems used in ophthalmic applications, such ashyaluronic acid. The acrylamide polymers may have an average molecularweight in the range 1-13 million, preferably about 4-6 million. Theweight percent of the acrylamide polymer in the gel may be 2-5%,preferably 3.5-4.5%. Substituted acrylamide polymers, such as methyl andalkyl substituted polymers are also within the scope of the presentinvention.

For use in the anterior chamber of the eye, an acrylamide gel deliverysystem has the following characteristics: any products of thedissolution or degradation of the delivery matrix are nontoxic and donot clog the trabecular mesh work; the gel is optically transparent; andthe gel can be left in the anterior chamber without causing adverseclinical effects such as an unacceptable increase in ocular pressure.

It will be readily apparent to one skilled in the art that the desiredviscosity range may be achieved by varying the molecular weight andpercent concentration of the polymer in the formulation. For example, agel having a low viscosity may be achieved by using a low molecularweight polymer or a lower percent concentration or a combination of thetwo. A high viscosity gel may be achieved by using a higher molecularweight polymer and a higher percent concentration. Intermediateviscosities may be achieved by varying the molecular weight and percentconcentration accordingly.

The low viscosity solution may comprise 0.01-2.0% by weight polyacrylicacid having an molecular weight of about 100,000-4,000,000. In apreferred embodiment, the polymer is present at 0.05-0.5%. In anotherembodiment, this dilute viscous solution may comprise 2-40% by weight ofa polyoxyethylene-polyoxypropylene copolymer having an average molecularweight of 500-500,000. Preferably, the concentration is 2-20% and themolecular weight is 1,000-15,000. Alternatively, the dilute viscoussolution may comprise a cellulose polymer at 1-20% and having amolecular weight of about 80,000-240,000. It is preferred that theconcentration be in the range of 1-10%. In a further embodiment, thedilute viscous solution may comprise 0.5-5.0% by weight hyaluronic acidhaving an average molecular weight of about 500,000-8,000,000.Preferably, the concentration is 0.5-2.0% and the average molecularweight is 1,000,000-6,000,000. If the dilute viscous solution is to beused as eye drops, it is preferred that the viscosity be in the range1-100 cps. If it is used for other applications, such as soaking abandage, then any viscosity in the range 1.0-5,000 will be suitable.

Upon formulation, solutions are administered in a manner compatible withthe dosage formulation and in such amount as is therapeuticallyeffective to result in an improvement or remediation of the symptoms.The formulations are easily administered in a variety of dosage formssuch as ingestible solutions, drug release capsules, gel ointments andthe like. Some variation in dosage can occur depending on the conditionof the subject being treated. The person responsible for administrationcan, in any event, determine the appropriate dose for the individualsubject. Moreover, for human administration, preparations meetsterility, general safety and purity standards as required by FDA Officeof Biologics standards.

IV. Methods of Treatment Using the Lactoferrin Related Peptides

In certain aspects of the present invention, a peptide compositioncomprising lactoferrin related peptides or related-compounds thereof isadministered to treat a pathological condition, for example suchconditions, include, but are not limited to hyperproliferative disease,respiratory disease, cardiovascular disease, neurological condition,autoimmune disorder, infectious disease, gastrointestinal disorder,endocrine and/or metabolism disorder, hematological disorder, oculardisorder, integument disorder, pain and a wound.

It is envisioned that the immune system, whether local, systemic ormucosal, is enhanced by lactoferrin related peptides stimulatingcytokines and/or chemokines. Exemplary cytokines include interleukin-18and GM-CSF in the gastrointestinal tract, which are known to enhanceimmune cells or stimulate production of immune cells. For example,interleukin-18 enhances natural killer cells or T lymphocytes, which cankill bacteria infecting a wound. In specific embodiments, interleukin-18(IL-18) enhances CD4+, CD8+ and CD3+ cells. It is known by those ofskill in the art that IL-18 is a Th1 cytokine that acts in synergy withinterleukin-12 and interleukin-2 in the stimulation of lymphocyteIFN-gamma production. Other cytokines or chemokines may also be enhancedfor example, but not limited to IL-12, IL-1b, MIP-3α, MIP-1α orIFN-gamma. Other cytokines or enzymes may be inhibited for example, butnot limited to IL-2, IL-4, IL-5, IL-10, TNF-α, or matrixmetalloproteinases.

In further embodiments, cytokines, for example, interleukin-18 orgranulocyte/macrophage colony-stimulating factor, can stimulate theproduction or activity of immune cells. The immune cells include, butare not limited to T lymphocytes, natural killer cells, macrophages,dendritic cells, and polymorphonuclear cells. More specifically, thepolymorphonuclear cells are neutrophils and the T lymphocytes areselected from the group consisting of CD4+, CD8+ and CD3+ T cells.

Still further, it is envisioned that lactoferrin related peptidesstimulate production of MIP-3alpha from hepatocytes. Lactoferrin isknown to contribute to the defense systems of the body through itsanti-microbial properties. In addition, evidence suggests thatrecombinant human lactoferrin (rhLF) elicits a more general innate-likeimmune response when administered orally. The innate immune system isthe ‘first line of defense’ of the body against hostile environments andcomprise of a variety of effector and cellular mechanisms. This innateimmune response is initially likely mediated by the ‘detection system’of receptors known to be present on the surface of the gut epithelialcells, such as pattern recognition receptors, IL-1 receptor and general‘scavenger’ receptors. These receptors recognize and respond to specificstructural features of the presented molecules. As a result, variousintracellular signaling pathways may be initiated (e.g., NFκB, Wnt,etc.) that result in the overall orchestration of the cellular responseof the body to the prevailing biological situation (e.g., infection).RhLF and peptides derived from rhLF elicit a similar response of humanhepatocytes in vitro in terms of producing an important chemokine—namelyMIP-3-alpha. It is surmised that some general structural features thatare responsible for the biological response to rhLF are retained inpeptides that are structurally related to the parent molecule (rhLF).

The route of administration will vary, naturally, with the location andnature of the lesion, and include, for example parentally, orally ortopically. Parenteral administrations include, but are not limited tointradermal, transdermal, intravenous, intramuscular, intranasal,subcutaneous, percutaneous, intratracheal, intraocular, intraperitoneal,intratumoral, perfusion, lavage, direct injection U.S. Pat. Nos.6,613,308, 5,466,468, 5,543,158; 5,641,515; and 5,399,363 (eachspecifically incorporated herein by reference in its entirety).Alimentary administrations include, but are not limited to orally,buccally, rectally, or sublingually.

Treatment methods will involve treating an individual with an effectiveamount of a peptide composition as defined in the present invention. Aneffective amount is described, generally, as that amount sufficient todetectably and repeatedly ameliorate, reduce, minimize or limit theextent of a disease or its symptoms.

The effective amount or “therapeutically effective amounts” of thepeptide composition to be used are those amounts effective to producebeneficial results, particularly with respect to cancer treatment, inthe recipient animal or patient. Such amounts may be initiallydetermined by reviewing the published literature, by conducting in vitrotests or by conducting metabolic studies in healthy experimentalanimals. Before use in a clinical setting, it may be beneficial toconduct confirmatory studies in an animal model, preferably a widelyaccepted animal model of the particular disease to be treated. Preferredanimal models for use in certain embodiments are rodent models, whichare preferred because they are economical to use and, particularly,because the results gained are widely accepted as predictive of clinicalvalue.

As is well known in the art, a specific dose level of active compoundssuch as the peptide composition for any particular patient depends upona variety of factors including the activity of the specific compoundemployed, the age, body weight, general health, sex, diet, time ofadministration, route of administration, rate of excretion, drugcombination, and the severity of the particular disease undergoingtherapy. The person responsible for administration will determine theappropriate dose for the individual subject. Moreover, for humanadministration, preparations should meet sterility, pyrogenicity, andgeneral safety and purity standards as required by FDA Office ofBiologics standards.

A therapeutically effective amount of a peptide composition as atreatment vanes depending upon the host treated and the particular modeof administration. In one embodiment of the invention the dose range ofa peptide composition will be about 0.5 mg/kg body weight to about 500mg/kg body weight. The term “body weight” is applicable when an animalis being treated. When isolated cells are being treated, “body weight”as used herein should read to mean “total cell body weight”. The term“total body weight” may be used to apply to both isolated cell andanimal treatment. All concentrations and treatment levels are expressedas “body weight” or simply “kg” in this application are also consideredto cover the analogous “total cell body weight “and” total body weight”concentrations. However, those of skill will recognize the utility of avariety of dosage range, for example, 0.1 μg/kg body weight to 1 μg/kgbody weight, 1 μg/kg to 1 mg/kg body weight, 1 mg/kg body weight to 450mg/kg body weight, 2 mg/kg body weight to 400 mg/kg body weight, 3 mg/kgbody weight to 350 mg/kg body weight, 4 mg/kg body weight to 300 mg/kgbody weight, 5 mg/kg body weight to 250 mg/kg body weight, 6 mg/kg bodyweight to 200 mg/kg body weight, 7 mg/kg body weight to 150 mg/kg bodyweight, 8 mg/kg body weight to 100 mg/kg body weight, or 9 mg/kg bodyweight to 50 mg/kg body weight. Further, those of skill will recognizethat a variety of different dosage levels will be of use, for example,0.1 μg/kg, 1 μg/kg, 10 μg/kg, 100 μg/kg, 200 μg/kg; 300 μg/kg; 400μg/kg, 500 μg/kg, 600 μg/kg, 700 μg/kg, 800 μg/kg, 900 μg/kg, 1 mg/kg, 2mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 7.5 mg/kg, 10 mg/kg, 12.5 mg/kg, 15mg/kg, 17.5 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45mg/kg, 50 mg/kg, 60 mg/kg, 70 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, 120mg/kg, 140 mg/kg, 150 mg/kg, 160 mg/kg, 180 mg/kg, 200 mg/kg, 225 mg/kg,250 mg/kg, 275 mg/kg, 300 mg/kg, 325 mg/kg, 350 mg/kg, 375 mg/kg, 400mg/kg, 450 mg/kg, 500 mg/kg, 550 mg/kg, 600 mg/kg, 700 mg/kg, 750 mg/kg,800 mg/kg, 900 mg/kg, 1000 mg/kg, 1250 mg/kg, 1500 mg/kg, 1750 mg/kg,2000 mg/kg, 2500 mg/kg, and/or 3000 mg/kg. Of course, all of thesedosages are exemplary, and any dosage in-between these points is alsoexpected to be of use in the invention. Any of the above dosage rangesor dosage levels may be employed for the peptide composition of thepresent invention.

The treatments may include various “unit doses.” Unit dose is defined ascontaining a predetermined quantity of the therapeutic composition(lactoferrin related peptides or its related-compounds thereof)calculated to produce the desired responses in association with itsadministration, i.e., the appropriate route and treatment regimen. Thequantity to be administered, and the particular route and formulation,are within the skill of those in the clinical arts. Also of import isthe subject to be treated, in particular, the state of the subject andthe protection desired. A unit dose need not be administered as a singleinjection but may comprise continuous infusion over a set period oftime.

In certain embodiments, the composition is given in a single dose ormultiple doses. The single dose may be administered daily, or multipletimes a day, or multiple times a week, or monthly or multiple times amonth. In a further embodiment, the composition is given in a series ofdoses. The series of doses may be administered daily, or multiple timesa day, weekly, or multiple times a week, or monthly, or multiple times amonth.

A. Hyperproliferative Disease

The hyperproliferative disease, includes but is not limited toneoplasms. A neoplasm is an abnormal tissue growth, generally forming adistinct mass that grows by cellular proliferation more rapidly thannormal tissue growth. Neoplasms show partial or total lack of structuralorganization and functional coordination with normal tissue. These canbe broadly classified into three major types. Malignant neoplasmsarising from epithelial structures are called carcinomas, malignantneoplasms that originate from connective tissues such as muscle,cartilage, fat or bone are called sarcomas and malignant tumorsaffecting hematopoietic structures (structures pertaining to theformation of blood cells) including components of the immune system, arecalled leukemias, lymphomas and myelomas. A tumor is the neoplasticgrowth of the disease cancer. As used herein, a “neoplasm”, alsoreferred to as a “tumor”, is intended to encompass hematopoieticneoplasms as well as solid neoplasms. Examples of neoplasms include, butare not limited to melanoma, non-small cell lung, small-cell lung, lung,hepatocarcinoma, retinoblastoma, astrocytoma, glioblastoma, gum, tongue,leukemia, neuroblastoma, head, neck, breast, pancreatic, prostate,renal, bone, testicular, ovarian, mesothelioma, sarcoma, cervical,gastrointestinal, lymphoma, brain, colon, bladder, myeloma, or othermalignant or benign neoplasms.

Other hyperproliferative diseases include, but are not limited toneurofibromatosis, rheumatoid arthritis, Waginer's granulomatosis,Kawasaki's disease, lupus erythematosis, midline granuloma, inflammatorybowel disease, osteoarthritis, leiomyomas, adenomas, lipomas,hemangiomas, fibromas, vascular occlusion, restenosis, atherosclerosis,pre-neoplastic lesions, carcinoma in situ, oral hairy leukoplakia, orpsoriasis, and pre-leukemias, anemia with excess blasts, andmyelodysplastic syndrome.

Particular neoplasms of interest in the present invention include, butare not limited to hematopoietic neoplasms. For example, a hematopoieticneoplasm may include acute myelogenous leukemia, acute lymphoblasticleukemia, myelodysplastic syndrome, chronic myelomonocytic leukemia,juvenile myelomonocyte leukemia, multiple myeloma, chronic lymphocyticleukemia or other malignancy of hematologic origin.

In a preferred embodiment of the present invention, the peptidecompositions are administered in an effective amount to decrease,reduce, inhibit or abrogate the growth of a tumor. The amount may varyfrom about 0.1 μg to about 100 g of the lactoferrin composition.Preferably, the lactoferrin composition is orally administered in therange of 1 mg to 100 g per day, more preferably about 20 mg to about 10g per day with the most preferred dose being 4.5 g per day.Intravenously administered lactoferrin can be in the range of 0.1 μg toabout to 10 g per day, more preferably about 0.1 g to about 1 mg withthe most preferred dose being 250 mg per day. Preferably, a peptidecomposition is intratumorally administered in the range of 0.1 μg to 10g per day with the most preferred dose being 100 μg per day. Topically,the amount of lactoferrin related peptides may vary from about 1 μg toabout 100 g. Preferably, the topical gel, solution, capsule or tabletcomprises a concentration of about 0.01% to about 20% of lactoferrinrelated peptides. More preferably, the topical gel, solution, capsule ortablet may comprise a concentration of about 1% to about 8.5%lactoferrin related peptides.

In certain embodiments intratumoral administration of the compositionincludes intratumoral injection, electroporation, or surgical orendoscopic implantation. Intratumoral injection, or injection into thetumor vasculature is specifically contemplated for discrete, solid,accessible tumors. Local, regional or systemic administration also maybe appropriate.

The tumor being treated may not, at least initially, be resectable.Treatments with the lactoferrin composition may increase theresectability of the tumor due to shrinkage at the margins or byelimination of certain particularly invasive portions. Followingtreatments, resection may be possible. Additional treatments subsequentto resection will serve to eliminate microscopic residual disease at thetumor site.

Alternatively, the present invention may be used at the time of surgery,and/or thereafter, to treat residual or metastatic disease. For example,a resected tumor bed may be injected or perfused with a formulationcomprising the lactoferrin composition. The perfusion may be continuedpost-resection, for example, by leaving a catheter implanted at the siteof the surgery. Periodic post-surgical treatment is also envisioned.

Continuous administration also may be applied where appropriate, forexample, where a tumor is excised and the tumor bed is treated toeliminate residual, microscopic disease. Delivery via syringe orcatherization is preferred. Such continuous perfusion may take place fora period from about 1-2 hours, to about 6-12 hours, to about 12-24hours, to about 1-2 days, to about 1-2 weeks or longer following theinitiation of treatment. Generally, the dose of the therapeuticcomposition via continuous perfusion will be equivalent to that given bya single or multiple injections, adjusted over a period of time duringwhich the perfusion occurs. It was further contemplated that limbperfusion may be used to administer therapeutic compositions of thepresent invention, particularly in the treatment of melanomas andsarcomas.

Still yet, a further embodiment is a method of enhancing a mucosalimmune response in the gastrointestinal tract in a subject comprisingthe step of administering orally to said subject a peptide composition.It is also envisioned that the lactoferrin related peptide stimulatesinterleukin-18 following oral administration, which inhibitsangiogenesis and thereby has activity against tumor cells which aredependent on neovascularization.

A further embodiment of the present invention is a method of treating ahyperproliferative disease comprising the step of supplementing thesystemic immune system by increasing the amount of lactoferrin relatedpeptides in the systemic circulation. Preferably, the composition isadministered intravenously. It is envisioned that the lactoferrinrelated peptides stimulate interleukin-18 following intravenousadministration, which inhibits angiogenesis and thereby has activityagainst tumor cells which are dependent on neovascularization.

A further embodiment of the present invention is a method of treating ahyperproliferative disease comprising the step of supplementing a localor systemic immune system by increasing the amount of lactoferrin in thevicinity of the tumor. Vicinity of the tumor refers to the general areaof the tumor, for example the lactoferrin can be administered directlyinto or on the tumor, or in the general area of the tumor, but notdirectly into the tumor. The general area may include the margin area ornear or adjacent the margin area of the tumor. Preferably, thelactoferrin composition is administered intratumorally. It is envisionedthat the lactoferrin related peptides stimulate interleukin-18 followingintratumoral administration, which inhibits angiogenesis and thereby hasactivity against tumor cells which are dependent on neovascularization.

A further embodiment of the present invention is a method of treating ahyperproliferative disease comprising the step of supplementing a localor systemic immune system by increasing the amount of a lactoferrinrelated peptide in the skin in the vicinity of the tumor. Preferably,the composition is administered topically. As above, administration inthe vicinity of the tumor includes administration near or adjacent tothe margins of the tumor or directly in the margin area of the tumor. Itis envisioned that the lactoferrin related peptides stimulateinterleukin-18 and GM-CSF in the local tissue (e.g., keratinocytes),which enhances immune cells. It is envisioned that the lactoferrinrelated peptides stimulate interleukin-18 following intratumoraladministration, which inhibits angiogenesis and thereby has activityagainst tumor cells which are dependent on neovascularization.

In certain embodiments, it may be desirable to combine the peptidecomposition of the present invention with other agents effective in thetreatment of hyperproliferative disease, such as anti-cancer agents, orwith surgery. An “anti-cancer” agent is capable of negatively affectingcancer in a subject, for example, by killing cancer cells, inducingapoptosis in cancer cells, reducing the growth rate of cancer cells,reducing the incidence or number of metastases, reducing tumor size,inhibiting tumor growth, reducing the blood supply to tumor or cancercells, promoting an immune response against cancer cells or a tumor,preventing or inhibiting the progression of cancer, or increasing thelifespan of a subject with cancer. Anti-cancer agents include biologicalagents (biotherapy), chemotherapy agents, and radiotherapy agents. Moregenerally, these other compositions would be provided in a combinedamount effective to kill or inhibit proliferation of the cell. Thisprocess may involve administering the peptide composition of the presentinvention and the agent(s) or multiple factor(s) at the same time. Thismay be achieved by administering a single composition or pharmacologicalformulation that includes both agents, or by administering two distinctcompositions or formulations, at the same time, or at times close enoughso as to result in an overlap of this effect, wherein one compositionincludes the peptide composition and the other includes the secondagent(s).

Alternatively, the peptide composition of the present invention mayprecede or follow the other anti-cancer agent treatment by intervalsranging from minutes to weeks. In embodiments where the otheranti-cancer agent and peptide composition are administered or appliedseparately to the cell, one would generally ensure that a significantperiod of time did not expire between the time of each delivery, suchthat the agent and peptide composition would still be able to exert anadvantageously combined effect on the cell. In such instances, it iscontemplated that one may contact the cell with/administer bothmodalities within about 1-14 days of each other and, more preferably,within about 12-24 hours of each other. In some situations, it may bedesirable to extend the time period for treatment significantly,however, where several days (2, 3, 4, 5, 6 or 7) to several weeks (2, 3,4, 5, 6, 7 or 8) lapse between the respective administrations.

a) Chemotherapy

Cancer therapies also include a variety of chemical based treatments.Some examples of chemotherapeutic agents include without limitationantibiotic chemotherapeutics such as Doxorubicin, Daunorubicin,Adriamycin, Mitomycin (also known as mutamycin and/or mitomycin-C),Actinomycin D (Dactinomycin), Bleomycin, Plicomycin, plant alkaloidssuch as Taxol, Vincristine, Vinblastine, miscellaneous agents such asplatinum based agents (e.g., Cisplatin (CDDP)), etoposide (VP16), TumorNecrosis Factor, and alkylating agents such as, Carmustine, Melphalan(also known as alkeran, L-phenylalanine mustard, phenylalanine mustard,L-PAM, or L-sarcolysin, (a phenylalanine derivative of nitrogenmustard), Cyclophosphamide, Chlorambucil, Busulfan (also known asmyleran), taxane based agents (e.g., docetaxel) and Lomustine.

Some examples of other agents include, but are not limited to,Carboplatin, Procarbazine, Mechlorethamine, Irinotecan, Topotecan,Ifosfamide, Nitrosurea, Etoposide (VP16), Tamoxifen, Raloxifene,Toremifene, Idoxifene, Droloxifene, TAT-59, Zindoxifene, Trioxifene, ICI182,780, EM-800, Estrogen Receptor Binding Agents, Gemcitabinen,Navelbine, Farnesyl-protein transferase inhibitors, Transplatinum,5-Fluorouracil, hydrogen peroxide, and Methotrexate, Temazolomide (anaqueous form of DTIC), Mylotarg, Dolastatin-10, Bryostatin, or anyanalog or derivative variant of the foregoing.

b) Radiotherapeutic Agents

Radiotherapeutic agents and factors include radiation and waves thatinduce DNA damage for example, γ-irradiation, X-rays, UV-irradiation,microwaves, electronic emissions, radioisotopes, and the like. Therapymay be achieved by irradiating the localized tumor site with the abovedescribed forms of radiations. It is most likely that all of thesefactors effect a broad range of damage to DNA, the precursors of DNA,the replication and repair of DNA, and the assembly and maintenance ofchromosomes.

Dosage ranges for X-rays range from daily doses of 50 to 200 roentgensfor prolonged periods of time (3 to 4 weeks), to single doses of 2000 to6000 roentgens. Dosage ranges for radioisotopes vary widely, and dependon the half-life of the isotope, the strength and type of radiationemitted, and the uptake by the neoplastic cells.

c) Surgery

Approximately 60% of persons with cancer will undergo surgery of sometype, which includes preventative, diagnostic or staging, curative andpalliative surgery. Curative surgery is a cancer treatment that may beused in conjunction with other therapies, such as the treatment of thepresent invention, chemotherapy, radiotherapy, hormonal therapy, genetherapy, immunotherapy and/or alternative therapies.

Curative surgery includes resection in which all or part of canceroustissue is physically removed, excised, and/or destroyed. Tumor resectionrefers to physical removal of at least part of a tumor. In addition totumor resection, treatment by surgery includes laser surgery,cryosurgery, electrosurgery, and miscopically controlled surgery (Mohs'surgery). It is further contemplated that the present invention may beused in conjunction with removal of superficial cancers, precancers, orincidental amounts of normal tissue.

Upon excision of part of all of cancerous cells, tissue, or tumor, acavity may be formed in the body. Treatment may be accomplished byperfusion, direct injection or local application of the area with anadditional anti-cancer therapy. Such treatment may be repeated, forexample, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. Thesetreatments may be of varying dosages as well.

d) Other Biotherapy Agents

It is contemplated that other biological agents may be used incombination with the present invention to improve the therapeuticefficacy of treatment. These additional agents include, withoutlimitation, agents that affect the upregulation of cell surfacereceptors and GAP junctions, cytostatic and differentiation agents,inhibitors of cell adhesion, agents that increase the sensitivity of thehyperproliferative cells to apoptotic inducers, or other biologicalagents, as well as biotherapy such as for example, hyperthermia.

Hyperthermia is a procedure in which a patient's tissue is exposed tohigh temperatures (up to 106° F.). External or internal heating devicesmay be involved in the application of local, regional, or whole-bodyhyperthermia. Local hyperthermia involves the application of heat to asmall area, such as a tumor. Heat may be generated externally withhigh-frequency waves targeting a tumor from a device outside the body.Internal heat may involve a sterile probe, including thin, heated wiresor hollow tubes filled with warm water, implanted microwave antennae, orradiofrequency electrodes.

A patient's organ or a limb is heated for regional therapy, which isaccomplished using devices that produce high energy, such as magnets.Alternatively, some of the patient's blood may be removed and heatedbefore being perfused into an area that will be internally heated.Whole-body heating may also be implemented in cases where cancer hasspread throughout the body. Warm-water blankets, hot wax, inductivecoils, and thermal chambers may be used for this purpose.

Hormonal therapy may also be used in conjunction with the presentinvention. The use of hormones may be employed in the treatment ofcertain cancers such as breast, prostate, ovarian, or cervical cancer tolower the level or block the effects of certain hormones such astestosterone or estrogen and this often reduces the risk of metastases.

Adjuvant therapy may also be used in conjunction with the presentinvention. The use of adjuvants or immunomodulatory agents include, butare not limited to tumor necrosis factor; interferon alpha, beta, andgamma; IL-2 and other cytokines; F42K and other cytokine analogs; orMIP-1, MIP-1 beta, MCP-1, RANTES, and other chemokines.

e) Immunotherapy

Immunotherapeutics, generally, rely on the use of immune effector cellsand molecules to target and destroy cancer cells. The immune effectormay be, for example, an antibody specific for some marker on the surfaceof a tumor cell. The antibody alone may serve as an effector of therapyor it may recruit other cells to actually affect cell killing. Theantibody also may be conjugated to a drug or toxin (chemotherapeutic,radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) andserve merely as a targeting agent. Alternatively, the effector may be alymphocyte carrying a surface molecule that interacts, either directlyor indirectly, with a tumor cell target. Various effector cells includecytotoxic T cells and NK cells.

It is contemplated that vaccines that are used to treat cancer may beused in combination with the present invention to improve thetherapeutic efficacy of the treatment. Such vaccines include peptidevaccines or dendritic cell vaccines. Peptide vaccines may include anytumor-specific antigen that is recognized by cytolytic T lymphocytes.Yet further, one skilled in the art realizes that dendritic cellvaccination comprises dendritic cells that are pulsed with a peptide orantigen and the pulsed dendritic cells are administered to the patient.

Examples of tumor-specific antigens that are being used as vaccines inmelanoma include, but are not limited to gp 100 or MAGE-3. Theseantigens are being administered as peptide vaccines and/or as dendriticcell vaccines.

B. Wounds and Other Integument Disorders

1. Wounds

In accordance with the present invention, a peptide composition providedin any of the above-described pharmaceutical carriers is orally,topically, or parenterally administered to a subject suspected of orhaving a wound.

The present invention is designed for the treatment of any type ofwound, which includes, but is not limited to skin wound, internal wound,gastrointestinal wound, oral wound, bone wounds, ophthalmic wound,surgical wound, or any combination thereof. Wounds can be found on butnot limited to skin, internal organs, stomach and intestines(gastrointestinal), oral mucosa, and eye (ophthalmic wounds, e.g.,corneal ulcers, radiokeratotomy, corneal transplants, epikeratophakiaand other surgically induced wounds in the eye). Depending on theprocess that causes the wounds, wounds can also be classified as but arenot limited to incisional wounds, excisional wounds, diabetic ulcers,venous stasis ulcers, decubitus or pressure ulcers, chemical wounds, andburn wounds.

A further embodiment of the present invention is administering theinventive composition to treat skin wounds. Skin wounds further comprisebut are not limited to full-thickness wounds and partial-thicknesswounds. Full-thickness wounds involve the complete removal of epidermisand dermis to the depth of fascial planes or subcutaneous fat. In theloose-skinned species, the thin musculature of the panniculus carnosus,which firmly adheres to the base of the dermis, is usually removed aswell. In partial-thickness wounds a substantial amount of dermis, mostlyreticular, is left behind, and, more importantly, the bases of mostepidermal appendages (sebaceous and sweat glands, hair follicles) remainintact.

Yet further, a wound can be further defined as an acute wound. Acutewounds have a relatively rapid rate of healing, especially in healthysubjects. However, in the elderly or immunocompromised healing can beprolonged. Healing is also prolonged if the wound becomes infected.Preferred acute wounds that are to be treated with the presentcomposition include, but are not limited to partial-thickness burns,lacerations, bullet wounds or infected wounds.

A wound is also further defined as a chronic wound. Examples of chronicwounds or chronic ulcers include, but are not limited to diabeticulcers, venous stasis ulcers, decubitus or pressure ulcers. Yet further,chronic wounds can also include infected wounds. Chronic wounds arewounds that do not repair or do so extremely slowly, and show partial ortotal lack of structural organization and functional coordination withnormal tissue. Chronic wounds or chronic ulcers can be broadlyclassified into three major types: diabetic ulcers, venous stasisulcers, decubitus or pressure ulcers. Diabetic ulcers often occur on afoot. Chronic diabetic state and poor glucose control results in poorperipheral circulation and microcirculation due to progressivearteriosclerosis; neuropathic changes that result in an insensateextremity prone to trauma; and intrinsic defects in the wound healingprocess that may include reduced abundance and response to cellulargrowth factors. In the case of venous ulcers, venous hypertension causesdisturbed microcirculation and pathological changes of the capillaries,elevated persistent levels of pro-inflammatory cytokines and proteases.Fibroblast senesce and respond less to growth factors, which distributeunfavorably. Proteolytic enzymes and their inhibitors are imbalanced.Pressure ulcers occur when skin is under pressure without movement toallow blood flow for 8-12 hours.

In a preferred embodiment of the present invention, the inventivecomposition is administered in an effective amount to seal, to close, toimprove or to repair the wound. Also, it is envisioned that thecomposition of the present invention can also decrease, reduce, orinhibit, bacterial infections of the wound, which aid in the healingprocess of a wound. Thus, one of skill in the art realizes thatdepending upon the wound type, location, health of the subject, etc.,the peptide composition of the present invention may be administered forany given period of time until the wound is healed at least by 5%, 10%,20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%or any range in between.

For topical administration, the gel formulation of the present inventionmay be used to coat fibers of an absorbent gauze dressing to form awound healing bandage which may then be placed on a wound. The lowviscosity formulation is preferred for this use. The wound healingbandage may be prepared by soaking a gauze dressing with an aqueous gelsolution containing lactoferrin having wound healing activity. Thebandage can then be applied to the wound so that the coated fibers ofthe gauze contacts the wound and stimulate the rate of wound healing.

In those applications where the present invention is a gel that isapplied to an internal or incisional wound, it is preferred that the gelforming polymer be biodegradable. The naturally occurring polymers aregenerally biodegradable. Examples of these are collagen, theglycosaminoglycans, gelatin and starch. The cellulosics are notbiodegradable. The synthetic polymers such as the vinyl polymers are notdegradable. The biodegradability of the polymers described herein iswell known to those skilled in the art.

A further embodiment of the present invention is a method of treating awound comprising the step of supplementing the local immune system byincreasing the amount of a lactoferrin related peptide in the vicinityof the wound. Preferably, the peptide is administered topically to thewound.

Yet further, the present invention also provides a method of treating awound comprising the step of supplementing the systemic immune system byincreasing the amount of a lactoferrin related peptide in the systemiccirculation. Preferably, the lactoferrin is administered via aparenteral route, which includes, but is not limited to intramuscular,intravenous, intraperitoneal, intraocular, intraarticular or into asurgical field.

In further embodiments, the present invention provides a method ofenhancing the immune system of a subject suffering from a wound byadministering to the subject a peptide composition. Depending upon themode of administration, different arms of the immune system areenhanced. For example, topical administration of the composition resultsin enhancement of the local immune system, i.e., in the vicinity of thewound. Parenteral administration of the composition results inenhancement of the systemic immune system. Yet further, oraladministration of the composition results in enhancement of the mucosalimmune system, which can also result in systemic effects as well. It isfurther contemplated that IL-18 or GM-CSF stimulate the production oractivity of cells involved in wound repair, for example, but not limitedto keratinocytes, endothelial cells, dendritic cells, fibroblasts, andmyofibroblasts. Yet further, it is envisioned that lactoferrin relatedpeptides inhibit the production of TNF-alpha, which may lead to excessinflammation and tissue destruction for example by stimulating theproduction of metalloproteinases.

It is further envisioned that supplementing the local immune system in asubject by administering topically a therapeutically effective amount ofthe inventive composition in the vicinity of the wound can result in thekilling of bacteria infecting the wound. Still further, topicaladministration of a composition comprising a lactoferrin peptide relatedcompound may stimulate the production of a cytokine or a chemokine.Cytokines, for example, interleukin-18 or granulocyte/macrophagecolony-stimulating factor, can also stimulate the production or activityof cells involved in wound repair. The cells involved in wound repairinclude, but are not limited to keratinocytes, endothelial cells,fibroblasts, dendritic cells, and myofibroblasts. The inhibition ofTNF-alpha further inhibits the migration and maturation of dendriticcells and the production of metalloproteinases. The dendritic cells canbe Langerhans cells.

In order to increase the effectiveness of the composition of the presentinvention, it may be desirable to combine the composition of the presentinvention with other agents effective in the treatment of wounds, suchas growth factors, skin replacement therapy, enzymatic and surgicaldebridement, moist wound dressings, cleansers, antibiotics. Such woundhealing agents are capable of negatively affecting a wound in a subject,for example, by enhancing the growth rate of skin cells, augmenting theblood supply to skin cells, promoting an immune response againstbacteria infecting the wound, killing bacteria, cleaning ischemictissue, promoting the closure of the wound. More generally, these otherwound healing agents are provided in a combined amount effective topromote the healing of a wound. This process may involve administeringthe composition of the present invention and the agent(s) or multiplefactor(s) at the same time. This may be achieved by administering asingle composition or pharmacological formulation that includes bothagents, or by administering two distinct compositions or formulations,at the same time, or at times close enough so as to result in an overlapof this effect, wherein one composition includes the lactoferrincomposition and the other includes the second agent(s).

Alternatively, the composition of the present invention may precede orfollow the other wound healing agent treatment by intervals ranging fromminutes to weeks. In embodiments where the other wound healing agent andinventive composition are administered or applied separately to thewound, one would generally ensure that a significant period of time didnot expire between the time of each delivery, such that the agent andhuman lactoferrin composition would still be able to exert anadvantageously combined effect on the wound. In such instances, it iscontemplated that one may contact the wound with/administer bothmodalities within about 1-14 days of each other and, more preferably,within about 12-24 hours of each other. In some situations, it may bedesirable to extend the time period for treatment significantly,however, where several days (2, 3, 4, 5, 6 or 7) to several weeks (2, 3,4, 5, 6, 7 or 8) lapse between the respective administrations.

a) Growth Factors

Wound healing therapies include growth factor based treatments. Examplesinclude, but are not limited to Regranex™ (Becaplermin-BB gel),AuTolo-Gel (autologous activated platelet releasate), Procuren(autologous thrombin-induced platelet releasate). Growth factors actwithout limitation by promoting granulation or the formation of newhighly vascularized connective tissue; stimulating proliferation,differentiation and migration of epithelial cells, vascular endothelialcells and other skin cells; enhancing the production of collagen,collagenase, and extracellular matrix.

b) Skin Replacement Therapy

Examples include but are not limited to Apligraf (bilayered livingskin), Trancyte (Human fibroblast-derived temporary skin substitute),Dermagraft (permanent, one-layer skin substitute), Epicel (livingone-layer artificial skin), Integra (collagen-based skin regenerationtemplate), AlloDerm (single-layer artificial skin made from humancadavers), CCS (living, cultured, artificial skin).

c) Enzymatic and Surgical Debridement

Debridement is a process or procedure to clean ischemic or dead tissue.Enzymatic debriders include Accuzyme papain-urea debriding ointment andCollagenase Santyl. Surgical debridement refers to physical removal ofat least part of the ischemic or dead tissue in a wound. Debridement maybe repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1,2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12months. Enzymatic debridement treatments may be of varying dosages aswell. It is further contemplated that the present invention may be usedin conjunction with enzymatic or surgical debridement.

d) Dressings

Wound healing therapies include a variety of treatments based ondressings. Dressing categories include but are not limited to amorphoushydrogels, hydrogel sheets, absorptives, alginates, biological andsynthetic dressings, collagens, composites, contact layers, elasticgauzes, foams, gauzes and non-woven dressings, hydrocolloids,impregnated dressings, silicone gel sheets, silver dressings,transparent films, wound fillers

e) Cleansers

Examples include but are not limited to Biolex, Lamin, Wound WashSaline, Techni-Care, CarraKlenz, DiaB Klenz, MicroKlenz, RadiaCareKlenz, UltraKlenz, Comfee Sea-Clens, Optipore Sponge, Saf-Clens,Shur-Clens, Dermagran, DermaKlenz, Dumex, Gene Klenz, GRX, Allclenz,Restore, Hyperion, Medi Tech, Skintegrity, MPM Antimicrobial,ClinsWound, Septicare, Lobana Saline.

f) Antimicrobials

Examples of include but are not limited to Sulfamylon Cream, ThermazeneCream (1% silver sulfadiazine), cadexomer-iodine pads or gel. Examplesof intravenous antimicrobials include but are not limited toimipenem/cilastatin, β-lactam/β-lactamase inhibitors(ampicillin/sulbactam, piperacillin/tazobactam), and broad-spectrumcephalosporins (cefoxitin, ceftizoxime, ceftazidime). Other examplesinclude, but are not limited to Bensal HP, Barri-Care, Care-Creme,Formula Magic, Baza, Micro-Guard, Ca-Rezz, Diabet-X products, MitrazolPowder, PiercingCare, Triple Care products, and various antifungalcreams and powders.

g) Compression

Dynamic compression examples, include pumps and sleeves such as but notlimited to ArtAssist, ArterialFlow, EdemaFlow, PulStar, Circulator Boot,Flowplus, Flowpress, Flowtron. Static compression include but are notlimited to leg wrappings, gloves, socks, leg wears, leg supports, armsleeves, stasis pads, compression hosieries, non-elastic bands, highcompression bandages, zinc impregnated bandages, elastic bandages.

h) Oxygen Therapy

Examples of systemic hyperbaric oxygen therapy include but are notlimited to compartments for one patient to lay down, for one patient tosit up to 25 degree angle, for one patient to sit up to 90 degree angle,for more than one patient to be treated simultaneously. Examples oftopical hyperbaric oxygen therapy include but are not limited todisposable topical hyperbaric oxygen systems for extremity ulcers,disposable topical hyperbaric oxygen systems for decubitis, post-op andtrauma wounds.

i) Hydrotherapy, Electric Therapy

Examples include but are not limited to dry hydrotherapy machines;non-contact thermal wound care systems for use on partial- andfull-thickness wounds that maintain warmth and humidity in the woundarea; systems that provide non-thermal, pulsed high frequency, high peakpower, electromagnetic energy to treat edema and pain in acute andchronic wounds; systems that use controlled, localized negative pressureand support for moist wound healing; pulsatile irrigators withcontrollable pressures below 15 psi for site-specific treatment ofvarious wounds with variety of tips; various wound irrigation andwhirlpool systems.

j) Nutritional Therapy Products

Examples include but are not limited to isotonic, high-protein,fiber-containing tube feedings to support wound healing; high-protein,cholesterol-free nutritional supplements.

k) Cohesives, Glues, Sealants, Patches

Examples include but are not limited to Dermabond, CoStasis, CoSeal,BioGlue, FibRx, FocalSeal, FloSeal, AutoSeal, Indermil, Syvek,LiquiSheild, LiquiBand, Quixil, CryoSeal, VIGuard Fibrin Sealant, andvarious tapes, closures, and securement products.

l) Topical Wound Healing Promoters

Examples include but are not limited to topical aerosols which stimulatethe capillary bed of chronic wounds; skin protectants with zinc-nutrientformulations; topical gels to help scars feel softer and smoother;hydrophilic ointments that cleanse degraded proteins, promote healthygranulation, control local inflammation and reduce wound odors;oil-and-water wound dressing emulsions that selectively recruitmacrophages.

m) Other Biotherapy Agents

Adjuvant therapy may also be used in conjunction with the presentinvention. The use of adjuvants or immunomodulatory agents include, butare not limited to tumor necrosis factor; interferon alpha, beta, andgamma; IL-2 and other cytokines; F42K and other cytokine analogs; orMIP-1, MIP-1beta, MCP-1, RANTES, and other chemokines.

2. Psoriasis

In accordance with the present invention, a peptide composition providedin any of the above-described pharmaceutical carriers is orally,topically, or parenterally administered to a subject suspected of orhaving psoriasis.

Psoriasis is a chronic inflammatory skin disorder that undergoesrepeated relapse and remission and affects one to three percent of theworld's population. The disease can begin at any age and has its peakappearance in the third decade. Its severity, course, and remissions areunpredictable. (See, T. P. Habif, in: Clinical Dermatology, Mosby, Mo.(1996) and The Textbook of Medicine edts: J. B. Wyngaarden and L. H.Smith, W., W. B. Saundres Company, Philadelphia (1985)).

Patients with psoriasis can be divided into two groups, those with agenetic predisposition and those that respond to epigenetic, i.e.environmental factors. The most common form is chronic plaque psoriasisand is characterized by hyperplasia of the epidermis and inflammation ofthe dermis and epidermis. These changes arise due to activation of aT-lymphocyte cell mediated dermal immune reaction in regional lymphnodes in response to unidentified antigenic stimuli. The activatedT-cells cause keratinocyte cells to proliferate and assume a psoriasisphenotype. The inflammatory reaction is caused by proinflammatorycytokine proteins that are induced in response to the environmentalstimulus and results in infiltration of the dermis and epidermis byinflammatory white blood cells.

The present invention provides a method of enhancing the immune systemof a subject suffering from psoriasis by administering to the subject apeptide composition. Depending upon the mode of administration,different arms of the immune system are enhanced. For example, topicaladministration of the composition results in enhancement of the localimmune system, i.e., in the vicinity of the wound. Parenteraladministration of the composition results in enhancement of the systemicimmune system. Yet further, oral administration of the compositionresults in enhancement of the mucosal immune system, which can alsoresult in systemic effects as well.

Psoriasis has been shown to be caused by inappropriate local immuneresponses which involve the de novo production of TNF-α by keratinocytes(Gilhar et al., 1996, Clin. Exp. Immunol. 106: 134-142). Accordingly, inone embodiment, the present invention provides compositions thatdirectly interfere with the production of TNF-α by keratinocytes, thuspreventing the inappropriate local immune response causing psoriasis.More specifically, the present invention provides novel pharmaceuticalcompositions comprising a lactoferrin related peptide product for thetreatment of psoriasis. In a preferred embodiment, the compositions areformulated for topical application in about 0.5% to about 5% carrier. Inalternative embodiments, the compositions are formulated for intradermalinjections.

In order to increase the effectiveness of the composition of the presentinvention, it may be desirable to combine the composition of the presentinvention with other agents effective in the treatment of psoriasis,such as anti-inflammatory agents. This process may involve administeringthe composition of the present invention and the agent(s) or multiplefactor(s) at the same time. This may be achieved by administering asingle composition or pharmacological formulation that includes bothagents, or by administering two distinct compositions or formulations,at the same time, or at times close enough so as to result in an overlapof this effect, wherein one composition includes the lactoferrincomposition and the other includes the second agent(s).

Alternatively, the composition of the present invention may precede orfollow the other anti-inflammatory agent treatment by intervals rangingfrom minutes to weeks. In embodiments where the other anti-inflammatoryagent and inventive composition are administered or applied separatelyto the anti-inflammatory, one would generally ensure that a significantperiod of time did not expire between the time of each delivery, suchthat the agent and the inventive composition would still be able toexert an advantageously combined effect on the psoriasis.

Typical anti-inflammatory agents are topical agents, for example, butnot limited to are corticosteroids (hydrocortisone and analogs).Systemic corticosteroids, for example, can induce prompt resolution ofpsoriatic lesions, but suppression requires ever-increasing doses.

C. Respiratory Disorders

In accordance with the present invention, the composition provided inany of the above-described pharmaceutical carriers is orallyadministered to a subject suspected of or having a respiratory disorder.The respiratory disorder can be an allergic or non-allergic respiratorydisorder.

According to the invention, an allergic or non-allergic respiratorydisorder includes asthma, emphysema, bronchitis, chronic obstructivepulmonary disease, sinusitis and allergic rhinitis. In specificembodiments, the respiratory disorder is characterized by increasedresponsiveness of the trachea and bronchi to various stimuli, i.e.,allergens, resulting in widespread narrowing of the airways.

The composition is administered in an effective amount to decrease,reduce, inhibit or abrogate the obstruction of the airway or thehyperresponsiveness of the respiratory system to the allergen or otherstimuli. The amount of lactoferrin related peptide in the compositionmay vary from about 1 mg to about 100 g. Preferably, the compositionthat is orally administered contains the range of 1 mg to 10 g ofpeptide per day, more preferably, 10 mg to about 1 g. More preferably,the composition of the present invention also contains metal chelators.A preferred metal chelator is EDTA. More preferably, the compositionthat is orally administered contains the ratio of 1:10,000 to about 2:1EDTA to lactoferrin related peptide.

A further embodiment is a method of enhancing a mucosal immune responsein the gastrointestinal tract in a subject comprising the step ofadministering orally to said subject the composition of the presentinvention. It is envisioned that oral administration of lactoferrinrelated peptides stimulate IL-18 in the lungs, sinuses or systemically.Yet further, it is contemplated that an enhancement of lactoferrinrelated peptides can reduce the infiltration of inflammatory cells intothe lung.

In order to increase the effectiveness of administration of thecomposition of the present invention, it is desirable to combine thesecompositions with an additional agent. For example, known asthma agentsare used in combination with the composition of the present invention.Exemplary agents known to treat asthma include mast cell degranulationagents (i.e., Cromylyn sodium or Nedocromil sodium), leukotrieneinhibitors (i.e., Monteleukast sodium, Zafirlukast, or Pranlukasthydrate), corticosteroids (i.e., Beclomethasone, Budesonide,Ciclesonide, Hydrolysable glucocorticoid, Triamcinolone acetonide,Flunisolide, Mometasone furoate, or Fluticasone propionate),β-Antagonists (i.e., Albuterol, Bambuterol, Formoterol, Salbutamol,Terbutaline sulfate, or Salmeterol), IgE binding inhibitors (i.e.,Omalizumab), Adenosine A2 agonists, Anti-CD23 antibody, E-Selectinantagonists, P-Selectin antagonists, L-Selectin antagonists, interleukininhibitors/monoclonal antibodies, pulmonary surfactants, neurokininantagonists, NF-Kappa-B inhibitors, PDE-4 inhibitors (i.e., Cilomilast,or Roflumilast), Thromboxan A2 inhibitors (i.e., Ramatroban, orSeratrodast), tryptase inhibitors, VIP agonists or antisense agents.

Yet further, the agent is a known agent used to treat allergic rhinitis.Exemplary agents include, but are not limited to H1 antihistamines i.e.,terfendine or astemizole; alpha-adrenergic agents; and glucocorticoids,i.e., beclomethasone or flunisolide.

In addition, the agent is a known agent to treat sinusitis, morespecifically, chronic sinusitis. Exemplary agents/therapies include, butare not limited to surgery (e.g., enlarging a sinus passage, removeobstructing bone or nasal polyps, mucosal stripping, removal ofsinuses); corticosteroids (e.g., oral, intranasal, nebulized, orinhaled); antibiotics (e.g., oral, intranasal, nebulized, inhaled orintravenous); anti-fungal agents; salt-water nasal washes and mistsprays; anti-inflammatory agents; decongestants (oral or nasal);guaifenesin; potassium iodide; leukotriene inhibitors (e.g.,monteleukast); mast cell degranulating agents; topical moisturizingapplications (e.g., nasal sprays or gels which may contain moisturizingagents such as propylene glycol or glycerin); hot air inhalation;mechanical devices to aid in breathing; enzymatic cleansers (e.g.,papaya enzymes); and antihistamine sprays.

Still further, the agent is a known agent to treat COPD or chronicbronchitis or emphysema. Exemplary agents/interventions include, but arenot limited to oxygen; bronchodilator drugs [e.g., short and long actingbeta-2 stimulants, anticholinergics (e.g., ipratoprium bromide,theophylline compounds or a combination), steroids (topical or oral), ormucolytic agents (e.g., ambroxol, ergosterin, carbocysteine, iodinatedglycerol)]; antibiotics; anti-fungals; moisterization by nebulization;anti-tussives; respiratory stimulants (e.g., doxapram, almitrinebismesylate); surgery (e.g., bullectomy, lung volume reduction surgery,lung transplantation); and alpha 1 antitrypsin administration.

The composition of the present invention may precede, be co-current withand/or follow the other agent(s) by intervals ranging from minutes toweeks. In embodiments where the composition of the present invention,and other agent(s) are applied separately to a cell, tissue or organism,one would generally ensure that a significant period of time did notexpire between the time of each delivery, such that the composition andagent(s) would still be able to exert an advantageously combined effecton the cell, tissue or organism.

Various combination regimens of the composition and one or more agentsare employed. One of skill in the art is aware that the composition ofthe present invention and agents can be administered in any order orcombination.

D. Infectious Diseases

In certain embodiment of the invention, the present invention is usefulfor the treatment and/or prevention of infectious disease. Infectiousdiseases include infections of viral etiology such as HIV, West NileVirus, influenza, Herpes, viral hepatitis, Epstein Bar, polio, viralencephalitis, measles, chicken pox, Papilloma virus etc.; or infectionsof bacterial etiology such as bacterial meningitis, paramenigealinfections, septic thrombophlebitis, pneumonia, tuberculosis,myocarditis, bacteremia, syphilis, etc.; or infections of parasiticetiology such as malaria, trypanosomiasis, leishmaniasis, candidiasis,trichomoniasis, amoebiasis, etc.

1. Viral

The present invention can have applications therefore in the preventionand treatment of viral diseases. The following pathogenic viruses whichare mentioned by way of example, influenza A, B and C, parainfluenza,paramyxoviruses, Newcastle disease virus, respiratory syncytial virus,measles, mumps, adenoviruses, adenoassociated viruses, parvoviruses,Epstein-Barr virus (EBV), rhinoviruses, coxsackieviruses, echoviruses,reoviruses, rhabdoviruses, lymphocytic choriomeningitis, coronavirus,polioviruses, herpes simplex (HSV), human immunodeficiency viruses(HIV), cytomegaloviruses, papillomaviruses, human papillomavirus (HPV),hepatitis B virus (HBV), hepatitis C virus (HCV), varicella-zoster,poxviruses, rubella, rabies, picornaviruses, rotavirus and Kaposiassociated herpes virus.

2. Bacterial

In addition to the viral diseases mentioned above, the present inventionis also useful in the prevention, inhibition, or treatment of bacterialinfections. The following bacteria are mention by way of example,including, but not limited to, serotypes of pneumococci, streptococcisuch as S. pyogenes, S. agalactiae, S. equi, S. canis, S. bovis, S.equinus, S. anginosus, S. sanguis, S. salivarius, S. mitis, S. mutans,other viridans streptococci, peptostreptococci, other related species ofstreptococci, enterococci such as Enterococcus faecalis, Enterococcusfaecium, Staphylococci, such as Staphylococcus epidermidis,Staphylococcus aureus, particularly in the nasopharynx, Hemophilusinfluenzae, pseudomonas species such as Pseudomonas aeruginosa,Pseudomonas pseudomallei, Pseudomonas mallei, brucellas such as Brucellamelitensis, Brucella suis, Brucella abortus, Bordetella pertussis,Neisseria meningitidis, Neisseria gonorrhoeae, Moraxella catarrhalis,Corynebacterium diphtheriae, Corynebacterium ulcerans, Corynebacteriumpseudotuberculosis, Corynebacterium pseudodiphtheriticum,Corynebacterium urealyticum, Corynebacterium hemolyticum,Corynebacterium equi, etc. Listeria monocytogenes, Nocordia asteroides,Bacteroides species, Actinomycetes species, Treponema pallidum,Leptospirosa species and related organisms. The invention may also beuseful against gram negative bacteria such as Klebsiella pneumoniae,Escherichia coli, Proteus, Serratia species, Acinetobacter, Yersiniapestis, Francisella tularensis, Enterobacter species, Bacteriodes andLegionella species and the like.

3. Fungal Infections

Yet further, either fungal and other mycotic pathogens or cells infectedwith fungal or other mycotic pathogens may be used in the presentinvention to prevent and/or treat diseases, ranging from mycosesinvolving skin, hair, or mucous membranes, such as, but not limited to,Aspergillosis, Black piedra, Candidiasis, Chromomycosis, Cryptococcosis,Onychomycosis, or Otitis externa (otomycosis), Phaeohyphomycosis,Phycomycosis, Pityriasis versicolor, ringworm, Tinea barbae, Tineacapitis, Tinea corporis, Tinea cruris, Tinea favosa, Tinea imbricata,Tinea manuum, Tinea nigra (palmaris), Tinea pedis, Tinea unguium,Torulopsosis, Trichomycosis axillaris, White piedra. Fungal and mycoticpathogens that can be used in the present invention include, but are notlimited to, Absidia spp., Actinomadura madurae, Actinomyces spp.,Allescheria boydii, Alternaria spp., Anthopsis deltoidea, Apophysomyceselegans, Arnium leoporinum, Aspergillus spp., Aureobasidium pullulans,Basidiobolus ranarum, Bipolaris spp., Blastomyces dermatitidis, Candidaspp., Cephalosporium spp., Chaetoconidium spp., Chaetomium spp.,Cladosporium spp., Coccidioides immitis, Conidiobolus spp.,Corynebacterium tenuis, Cryptococcus spp., Cunninghamella bertholletiae,Curvularia spp., Dactylaria spp., Epidermophyton spp., Epidermophytonfloccosum, Exserophilum spp., Exophiala spp., Fonsecaea spp., Fusariumspp., Geotrichum spp., Helminthosporium spp., Histoplasma spp.,Lecythophora spp., Madurella spp., Malassezia furfur, Microsporum spp.,Mucor spp., Mycocentrospora acerina, Nocardia spp., Paracoccidioidesbrasiliensis, Penicillium spp., Phaeosclera dematioides,Phaeoannellomyces spp., Phialemonium obovatum, Phialophora spp., Phomaspp., Piedraia hortai, Pneumocystis carinii, Pythium insidiosum,Rhinocladiella aquaspersa, Rhizomucor pusillus, Rhizopus spp., Saksenaeavasiformis, Sarcinomyces phaeomuriformis, Sporothrix schenckii,Syncephalastrum racemosum, Taeniolella boppii, Torulopsosis spp.,Trichophyton spp., Trichosporon spp., Ulocladium chartarum, Wangielladermatitidis, Xylohypha spp., and Zygomyetes spp.

In addition, the invention may prove useful in controlling protozoan ormacroscopic infections by organisms such as Cryptosporidium, Isosporabelli, Toxoplasma gondii, Trichomonas vaginalis, Cyclospora species, forexample, and for Chlamydia trachomatis and other Chlamydia infectionssuch as Chlamydia psittaci, or Chlamydia pneumoniae, for example.

4. Bacteremia and Septic Shock

In accordance with the present invention, the composition provided inany of the above-described pharmaceutical carriers is administered to asubject suspected of or having bacteremia, sepsis, septic shock orsequelae. These conditions could be caused by gram-negative,gram-positive bacteria or other infectious agents such as Candida in anyfoci of the body and are at a risk of developing into or have developedinto a systemic inflammatory response syndrome.

Yet further, bacteremia may be caused by surgical manipulation ofinfected oral tissues or routine dental manipulations; catheterizationof an infected lower urinary tract; incision and drainage of an abscess;and colonization of indwelling devices, especially IV and intracardiaccatheters, urethral catheters, and ostomy devices and tubes. The primarysite of infection is usually in the lungs, in the GU or GI tract, or insoft tissues including the skin in patients with decubitus ulcer. Inchronically ill and immunocompromised subjects, gram-negative bacteremiaoccurs more commonly, than in a healthy subject. Additionally, theseimmunocompromised subjects may develop bloodstream infections caused byaerobic bacilli, anaerobes, and fungi.

Predisposing factors for septic shock include diabetes mellitus;cirrhosis; leukopenic states, especially those associated withunderlying neoplasms or treatment with cytotoxic agents; antecedentinfection in the urinary, biliary, or GI tracts; invasive devices,including catheters, drainage tubes, and other foreign materials; andprior treatment with antibiotics, corticosteroids, or ventilatorydevices. Septic shock occurs more often in newborns, subjects >35 yr,pregnant women, and those seriously immunocompromised by underlyingdiseases or iatrogenic complications of treatment.

According to the invention, the above-described method is used for theprophylaxis of bacteremia, sepsis, septic shock, related conditions ortheir consequences. In specific embodiments, the disorder ischaracterized by a risk of endotoxemia resulting from the use ofantibiotic and the subsequent release of endotoxin, as well aspositively identified bacteremia.

A person at risk for developing bacteremia, sepsis, septic shock and/orrelated conditions is a person that is considered to beimmunocompromised and/or chronically ill. The immunocompromised subject,who is, at the time of bacterial exposure, has a pre-existing conditionthat reduces one or more mechanisms for normal defense againstinfection. The immunocompromised condition may be due to a defect ordysfunction of the immune system or to other factors that heightensusceptibility to infection, for example immunosuppressive agents. Thus,prophylatically, it is envisioned that the lactoferrin composition canreduce any of the following: the levels of circulating bacteria, therisk of the subject developing sepsis, septic shock, organ failure, anddecrease the morbidity and mortality associated with bacteremia.

In a preferred embodiment of the present invention, the composition isadministered in an effective amount to decrease, reduce, inhibit orabrogate the risk of developing bacteremia and minimizing the effects ofalready existing bacteremia, sepsis, septic shock or related conditions.The amount of lactoferrin related peptides in the composition may varyfrom about 1 mg to about 100 g.

The guiding principle in the use of lactoferrin related peptides is toadminister the treatment at the earliest signs of bacteremia, sepsis orseptic shock being developed to attenuate the development of bacteremiaand to reduce the extent of organ damage that results from sepsis andseptic shock. The improvement is any observable or measurableimprovement. Thus, one of skill in the art realizes that a treatment mayimprove the patient or subject's condition, but may not be a completecure of the disease. In certain aspects, the composition is administeredin an effective amount to decrease, reduce, inhibit or abrogate levelsof bacteria in circulation. In further aspects, an improvement canconsist of any of the following, for example, decrease in the levels ofcirculating bacteria, attenuating the development of sepsis, attenuatingthe development of septic shock, attenuating the development of organfailure, decreasing morbidity associated with bacteremia and decreasingmortality (death) associated with bacteremia. Thus, after administrationof lactoferrin related peptides, if any of the above conditions improve,then the amount of lactoferrin related peptides are considered to be aneffective amount. Yet further, administration of lactoferrin relatedpeptides will also attenuate the development of sepsis, septic shock andother conditions related thereto.

In certain aspects, the composition is administered in an effectiveamount to decrease, reduce, inhibit or abrogate the severity of sepsisor septic shock. In further aspects, an improvement can consist of anyof the following, for example, decreasing mortality, decreasingmorbidity, attenuating the development organ failure, decreasing days ofhospitalization, decreasing or eliminating days of intensive care suchas in an intensive care unit, decreasing or eliminating the use ofsupportive care such as a mechanical ventilator or decreasing theincidence of sequelae such as ARDS. Survival in patients with organfailure at baseline and prevention and reversal of organ failure arealso evaluated. Thus, after administration of lactoferrin relatedpeptides, if any of the above conditions improve, then the amount oflactoferrin related peptides are considered to be an effective amount.

In certain aspects, the composition is administered in an effectiveamount to decrease, reduce, inhibit or abrogate the severity of ALI orARDS. In further aspects, an improvement can consist of any of thefollowing, for example, decrease in mortality, attenuating thedevelopment organ failure, decreasing days of hospitalization,decreasing or eliminating days of intensive care such as in an intensivecare unit, or decreasing or eliminating the use of supportive care suchas a mechanical ventilator or PaO₂/FiO₂ ratios. Thus, afteradministration of lactoferrin related peptides, if any of the aboveconditions improve, then the amount of are considered to be an effectiveamount.

In order to increase the effectiveness of the composition, it may bedesirable to combine these compositions and methods of the inventionwith a known agent effective in the treatment or prevention ofbacteremia, sepsis, septic shock and related conditions, for exampleknown agents to treat bacterial infections, e.g., antibiotics, knownagents for the treatment of sepsis, e.g., Drotrecogin alfa (activated)and agents to treat inflammation. In some embodiments, it iscontemplated that a conventional therapy or agent, including but notlimited to, a pharmacological therapeutic agent may be combined with thecomposition of the present invention.

The composition of the present invention may precede, be co-current withand/or follow the other agent(s) by intervals ranging from minutes toweeks. In embodiments where the composition of the present invention,and other agent(s) are applied separately to a cell, tissue or organism,one would generally ensure that a significant period of time did notexpire between the time of each delivery, such that the composition andagent(s) would still be able to exert an advantageously combined effecton the cell, tissue or organism.

Various combination regimens of the composition and one or more agentsare employed. One of skill in the art is aware that the composition ofthe present invention and agents can be administered in any order orcombination. In other aspects, one or more agents may be administeredsubstantially simultaneously, or within about minutes to hours to daysto weeks and any range derivable therein, prior to and/or afteradministering the composition.

Administration of the composition to a cell, tissue or organism mayfollow general protocols for the administration of cardiovasculartherapeutics, taking into account the toxicity, if any. It is expectedthat the treatment cycles would be repeated as necessary. In particularembodiments, it is contemplated that various additional agents may beapplied in any combination with the present invention.

Pharmacological therapeutic agents and methods of administration,dosages, etc. are well known to those of skill in the art (see forexample, the “Physicians Desk Reference”, Goodman & Gilman's “ThePharmacological Basis of Therapeutics”, “Remington's PharmaceuticalSciences”, and “The Merck Index, Eleventh Edition”, incorporated hereinby reference in relevant parts), and may be combined with the inventionin light of the disclosures herein. Some variation in dosage willnecessarily occur depending on the condition of the subject beingtreated. The person responsible for administration will, in any event,determine the appropriate dose for the individual subject, and suchindividual determinations are within the skill of those of ordinaryskill in the art.

Non-limiting examples of a pharmacological therapeutic agent that may beused in the present invention include an antimicrobial agent, ananti-sepsis agent, an anti-inflammatory agent, anantithrombotic/fibrinolytic agent, a blood coagulant, an antiarrhythmicagent, an antihypertensive agent, a vasopressor, or agents to treatmetabolic acidosis. In certain aspects of the present invention,antimicrobial agents, e.g., antibiotics are used in combination with thecomposition of the present invention.

a) Anti-Microbial Agents

In certain embodiments, an “antimicrobial agent” can be used incombination with the lactoferrin composition. An antimicrobial agent maycomprise an antibiotic, anti-fungal, and anti-viral agent.

Antibiotics inhibits the growth of microorganisms without damage to thehost. For example, the antibiotic may inhibit cell wall synthesis,protein synthesis, nucleic acid synthesis, or alter cell membranefunction. Classes of antibiotics that can possibly be used inconjunction with the peptide include, but are not limited to, macrolides(i.e., erythromycin), penicillins (i.e., nafcillin), cephalosporins(i.e., cefazolin), carbepenems (i.e., imipenem, aztreonam), otherbeta-lactam antibiotics, beta-lactam inhibitors (i.e., sulbactam),oxalines (i.e. linezolid), aminoglycosides (i.e., gentamicin),chloramphenicol, sulfonamides (i.e., sulfamethoxazole), glycopeptides(i.e., vancomycin), quinolones (i.e., ciprofloxacin), tetracyclines(i.e., minocycline), fusidic acid, trimethoprim, metronidazole,clindamycin, mupirocin, rifamycins (i.e., rifampin), streptogramins(i.e., quinupristin and dalfopristin) lipoprotein (i.e., daptomycin),polyenes (i.e., amphotericin B), azoles (i.e., fluconazole), andechinocandins (i.e., caspofungin acetate). Examples of specificantibiotics that can be used include, but are not limited to,erythromycin, nafcillin, cefazolin, imipenem, aztreonam, gentamicin,sulfamethoxazole, vancomycin, ciprofloxacin, trimethoprim, rifampin,metronidazole, clindamycin, teicoplanin, mupirocin, azithromycin,clarithromycin, ofloxacin, lomefloxacin, norfloxacin, nalidixic acid,sparfloxacin, pefloxacin, amifloxacin, gatifloxacin, moxifloxacin,gemifloxacin, enoxacin, fleroxacin, minocycline, linezolid,temafloxacin, tosufloxacin, clinafloxacin, sulbactam, clavulanic acid,amphotericin B, fluconazole, itraconazole, ketoconazole, and nystatin.Other examples of antibiotics, such as those listed in Sakamoto et al,U.S. Pat. No. 4,642,104 herein incorporated by reference will readilysuggest themselves to those of ordinary skill in the art.

Anti-viral agents can also be used in combination the peptidecomposition to treat and/or prevent a viral infection or disease. Suchanti-viral agents include, but are not limited to protease inhibitors(e.g., saquinavir, ritonavir, amprenavir), reverse transcriptaseinhibitors (e.g., azidothymidine (AZT), lamioridine (3TC),dideoxyinosine (ddI)), dideoxycytidine (ddC), zidovudine), nucleosideanalogs (e.g., acyclovir, penciclovir).

In certain embodiments, anti-fungal agents can be used in combinationwith the peptide composition to treat and/or prevent a fungal infection.Such anti-fungal agents include, amphotericin B (Amphocin®, Fungizone®),butoconazole (Femstat®), clotrimazole (Mycelex®, Gyne-Lotrimin®,Lotrimin®, Lotrisone®), fluconazole (Diflucan®), flucytosine (Ancobon®),griseofulvin (Fulvicin P/G®, Grifulvin V®, Gris-PEG®), itraconazole(Sporanox®), ketoconazole (Nizoral®), miconazole (Femizol-M®,Monistat®), nystatin (Mycostatin®), terbinafine (Lamisil®), terconazole(Terazol®), or tioconazole (Vagistat®).

b) Other Agents

Anti-sepsis agents include, but are not limited to Drotrecogin alfa(activated). Agents used for the treatment of ALI and ARDS include butare not limited to intra-pulmonary instillation of surfactants, andleukotriene modifiers. Anti-inflammatory agents include, but are notlimited to non-steroidal anti-inflammatory agents (e.g., naproxen,ibuprofen, celeoxib) and steroidal anti-inflammatory agents (e.g.,glucocorticoids).

Non-limiting examples of non-pharmacologic interventions that may beused in the present invention include supportive care such as organsupport in sepsis and septic shock and low tidal volume ventilationprotocols in ALI and ARDS.

E. Pain

In accordance with the present invention, the composition provided inany of the above-described pharmaceutical carriers is administered to asubject who has experienced pain which may be acute or chronic innature.

Causes of acute pain include, but are not limited to pain followingtrauma such as a blunt or sharp injury, bullet wounds, or surgery.

Causes of chronic pain include, but are not limited to pain associatedwith chronic diseases such as cancer, arthritis, inflammatory disease,chronic wounds, cardiovascular accidents, disorders of the spinal chordor central nervous system, and recovery from surgery.

In a preferred embodiment of the present invention, the composition isadministered in an effective amount to decrease, reduce, inhibit orabrogate pain. Thus, a subject is administered a therapeuticallyeffective amount of a lactoferrin composition so that the subject has animprovement in the parameters relating to pain including subjectivemeasures such as pain scores and grading and objective measuresincluding use of additional pain medications. The improvement is anyobservable or measurable improvement. Thus, one of skill in the artrealizes that a treatment may improve the patient condition, but may notbe a complete cure of the disease. The amount of lactoferrin in thecomposition may vary from about 1 ng to about 100 g each day.

In certain embodiments, the lactoferrin related peptide composition isadministered to a subject to modulate chronic or acute pain in thesubject. Modulation of pain is achieved by administering an effectiveamount of the composition to decrease, reduce, inhibit or abrogate pain.Specifically, it is contemplated that lactoferrin stimulates or enhancescytokine production or activity. For example, lactoferrin can enhancethe production or activity of TNF-α resulting in modulation of pain,acute or chronic, in the subject.

In order to increase the effectiveness of the composition, it may bedesirable to combine these compositions and methods of the inventionwith a known agent effective in the treatment of pain both directly andindirectly. In some embodiments, it is contemplated that a conventionaltherapy or agent, including but not limited to, a pharmacologicaltherapeutic agent, a non-pharmacological pain management techniques or acombination thereof, may be combined with the composition of the presentinvention.

The composition of the present invention may precede, be co-current withand/or follow the other agent(s) by intervals ranging from minutes toweeks. In embodiments where the composition of the present invention,and other agent(s) are applied separately to a cell, tissue or organism,one would generally ensure that a significant period of time did notexpire between the time of each delivery, such that the composition andagent(s) would still be able to exert an advantageously combined effecton the cell, tissue or organism.

Various combination regimens of the composition and one or more agentsare employed. One of skill in the art is aware that the composition ofthe present invention and agents can be administered in any order orcombination. In other aspects, one or more agents may be administeredwithin of from substantially simultaneously, about minutes to hours todays to weeks and any range derivable therein, prior to and/or afteradministering the composition.

Administration of the composition to a cell, tissue or organism mayfollow general protocols for the administration of pain therapeutics,taking into account the toxicity, if any. It is expected that thetreatment cycles would be repeated as necessary. In particularembodiments, it is contemplated that various additional agents may beapplied in any combination with the present invention.

a) Pharmacological Therapeutic Agents

Pharmacological therapeutic agents and methods of administration,dosages, etc. are well known to those of skill in the art (see forexample, the “Physicians Desk Reference”, Goodman & Gilman's “ThePharmacological Basis of Therapeutics”, “Remington's PharmaceuticalSciences”, and “The Merck Index, Eleventh Edition”, incorporated hereinby reference in relevant parts), and may be combined with the inventionin light of the disclosures herein. Some variation in dosage willnecessarily occur depending on the condition of the subject beingtreated. The person responsible for administration will, in any event,determine the appropriate dose for the individual subject, and suchindividual determinations are within the skill of those of ordinaryskill in the art.

Non-limiting examples of a pharmacological therapeutic agent that may beused in the present invention include non-steroidal anti-inflammatorydrugs (NSAIDS) (e.g., aspirin, indomethacin, ibuprofen, naproxen,acetaminophen, and ketoprofen); opioid analgesics (e.g., codeine,morphine, methadone, demerol, fentanyl, oxymorphone, and levorphanol),second generation NSAIDs (e.g., COX-2 inhibitors); and anti-depressantdrugs (e.g., tricyclin antidepressants, sedatives, tranquilizers,hypnotics, anti-histamines, and amphetamines).

b) Non-Pharmacological Pain Techniques

In certain aspects, a therapeutic agent may comprise anon-pharmacological pain management techniques Non-pharmacological painmanagement techniques are well known to those of skill in the art, andmay comprise, but are not limited to acupuncture or acupressure, localanesthesia/analgesia, regional anesthesia/analgesia (e.g., epidural orspinal analgesia/anesthesia), general anesthesia/analgesia (e.g.,intravenous anesthetics or opioid pump) and chiropractic.Non-pharmacological pain-reduction agents include, but are not limitedto devil's, capsaicin, menthold or L-phenylalanine.

F. Cardiovascular Diseases

In accordance with the present invention, the composition provided inany of the above-described pharmaceutical carriers is administered to asubject who has experienced or is at risk of developing cardiovasculardisease. Risk factors include, but are not limited to elevated levels ofcholesterol or CRP. One of skill in the art can determine the patientswho would potentially benefit from a therapeutic agent that would reducecirculating levels of total cholesterol or triglycerides orcardiovascular inflammation.

Cardiovascular diseases and/or disorders include, but are not limitedto, diseases and/or disorders of the pericardium, heart valves (e.g.,incompetent valves, stenosed valves, Rheumatic heart disease, mitralvalve prolapse, aortic regurgitation), myocardium (e.g., coronary arterydisease, myocardial infarction, heart failure, ischemic heart disease,angina) blood vessels (e.g., hypertension, arteriosclerosis, aneurysm)or veins (e.g., varicose veins, hemorrhoids). In specific embodiments,the cardiovascular disease is atherosclerosis.

In specific embodiments of the present invention, the peptidecomposition is administered to a subject suffering from or at risk fordeveloping atherosclerosis. Thus, it is envisioned that the peptidecomposition modulates or reduces the severity and/or incidence ofatherosclerosis.

Prophylactic treatment can be administered to those subjects at risk fordeveloping atherosclerosis. One risk factor is an atherogeniclipoprotein profile. For example, a ratio of serum cholesterol to highdensity lipoproteins of above 5:1 indicates a higher than average riskof developing atherosclerosis. Other factors indicating increased riskfor atherosclerosis include a serum cholesterol level of above 240mg/dl; a high density lipoprotein level below about 35 mg/dl; and a lowdensity lipoprotein level above about 160 mg/dl.

Another embodiment includes treating a human subject with an elevatedlevel of circulating total cholesterol or CRP according to the thenmedically established guidelines. It is contemplated that thecomposition of the present invention reduces or attenuates the levels ofcirculating total cholesterol, low density lipoproteins or very lowdensity lipoproteins. It is contemplated that the composition of thepresent invention can interfere with how cholesterol enters thecirculation either via absorption from food (exogenous pathway) orsynthesis by the liver (endogenous pathway).

Another embodiment includes treating a human subject havingdyslipidemia. Dyslipidemias are disorders of lipoprotein metabolism,including lipoprotein overproduction or deficiency. These disorders maybe manifested by elevation of the serum total cholesterol, low-densitylipoprotein (LDL) cholesterol and triglyceride concentrations, and adecrease in the high-density lipoprotein (HDL) cholesterolconcentration. Thus, it is envisioned that the composition of thepresent invention can reduce or abboragate the effects of lipoproteinmetabolism, such as dyslipidemia.

In a preferred embodiment of the present invention, the composition isadministered in an effective amount to decrease, reduce, inhibit orabrogate cardiovascular disease. Thus, a subject is administered atherapeutically effective amount of a lactoferrin composition so thatthe subject has an improvement in the parameters relating tocardiovascular disease including circulating levels of totalcholesterol, HDL, LDL, VLDL, triglycerides and C-reactive protein (CRP).The amount of lactoferrin in the composition may vary from about 1 ng toabout 20 g. Preferably, the composition that is orally administeredcontains the range of 0.5 g to 5 g of lactoferrin per day.

The improvement is any observable or measurable improvement. Thus, oneof skill in the art realizes that a treatment may improve the patientcondition, but may not be a complete cure of the disease. In certainaspects, the composition is administered in an effective amount todecrease, reduce, inhibit or abrogate excess amounts of cholesterollevels in circulation. A subject requires treatment for cholesterollevels based upon any of the following situations: LDL of 160 mg/ml orgreater; LDL of 130-159 mg/ml and also have two or more cardiovascularrisk factors; LDL of 100 mg/ml or greater in subjects with coronaryheart disease (CHD); triglycerides of 200 mg/dl or higher; totalcholesterol of 240 mg/dl or higher or HDL of less than 40 mg/dl. Thus,after administration of lactoferrin related compounds, if any of theabove conditions improve, then the amount is considered an effectiveamount to decrease, reduce, inhibit or abrogate cholesterol levels inthe circulation.

Another embodiment is a method of reducing vascular inflammation byadministering the composition of the present invention. Vascularinflammation can be tied to a number of the underlying processescontributing to atherosclerosis which include endothelial dysfunction,vascular proliferation and matrix alteration. Recent studies haveemphasized the involvement of inflammation in mediating all stages ofatherosclerosis. Vascular inflammation is thought to be a consequence ofdamage to the vascular endothelium and may also involve theproliferation of vascular smooth muscle cells (vsmcs). One precursor oflesion development in humans may be focal accumulation of vsmcs withinthe intima. In early atherosclerosis, vsmcs may contribute to thedevelopment of the atheroma through the production of pro-inflammatorymediators such as monocyte chemoattractant protein 1 and vascular celladhesion molecule, and through the synthesis of matrix moleculesrequired for the retention of lipoproteins. Inflammation of the vascularendothelium and proliferation of vsmcs may also impact the stability ofthe plaque through the formation of a firm fibrous cap. Indeed, inlipid-laden lesions in which the fibrous cap is thin and weak, there isevidence of vsmc apoptosis, especially at the “shoulder” region,associated with inflammation. In addition, the local inflammatory milieucan induce expression of collagenase and inhibit expression ofproteolytic inhibitors, thus rendering the fibrous cap weak andsusceptible to rupture. Lactoferrin, having known anti-inflammatoryproperties, may thus serve to inhibit the underlying processesassociated with the development of atherosclerosis.

In further embodiments, the composition may also reduce vascular spasmsor vascular hyper-reactivity. Vascular spasms are a sudden, brieftightening of a blood vessel, which can temporarily reduce blood flow totissues supplied by that vessel.

Still further, the composition may also promote endothelial integrity orhealing. Endothelia are the layer of cells lining the blood vessels.Endothelial dysfunction most commonly refers to impairment ofendothelium-dependent vasodilation and widespread abnormalities inendothelial integrity and homeostasis. It is believed that HDLs helpmaintain endothelial integrity, facilitate vascular relaxation, inhibitblood cell adhesion to vascular endothelium, reduce plateletaggregability and coagulation, and may favor fibrinolysis. The integrityor completeness of the endothelia lining of the vessels is important topreventing/treating the development of plaques and atherosclerosis.Thus, it is envisioned that the composition of the present inventionwill promote or modulate endothelial integrity or healing.

Yet further, another embodiment is a method of preventing acardiovascular disease in a subject at risk for developing acardiovascular disease comprising the step of administering to thesubject a peptide composition in an amount sufficient to result inprophylaxis of the cardiovascular disease in the subject. In preferredembodiments, the cardiovascular disease is atherosclerosis. It isenvisioned that the composition not only possess therapeutic benefitsfor those subjects suffering from cardiovascular diseases, but alsopossess prophylactic properties for those subjects at risk fordeveloping cardiovascular disease. A subject at risk may or may not becognizant of their disease state or potential disease state and may ormay not be aware that they are need of treatment. Thus, prophylatically,it is envisioned that the composition can reduce any of the following:the levels of circulating total cholesterol, low density lipoproteins(LDL), very low density lipoproteins (VLDL), levels of vascularinflammation, circulating C-reactive protein (CRP), triglycerides, andthe proliferation of vascular smooth muscle cells in the subject. Yetfurther, the composition may also increase the levels of circulatinghigh density lipoproteins (HDL).

In order to increase the effectiveness of the composition, it may bedesirable to combine these compositions and methods of the inventionwith a known agent effective in the treatment or prevention ofcardiovascular disease or disorder, for example known agents to treat orprevent atherosclerosis. In some embodiments, it is contemplated that aconventional therapy or agent, including but not limited to, apharmacological therapeutic agent, a surgical therapeutic agent (e.g., asurgical procedure) or a combination thereof, may be combined with thecomposition of the present invention.

The composition of the present invention may precede, be co-current withand/or follow the other agent(s) by intervals ranging from minutes toweeks. In embodiments where the composition of the present invention,and other agent(s) are applied separately to a cell, tissue or organism,one would generally ensure that a significant period of time did notexpire between the time of each delivery, such that the composition andagent(s) would still be able to exert an advantageously combined effecton the cell, tissue or organism.

Various combination regimens of the composition and one or more agentsare employed. One of skill in the art is aware that the composition ofthe present invention and agents can be administered in any order orcombination. In other aspects, one or more agents may be administeredsubstantially simultaneously, or within about minutes to hours to daysto weeks and any range derivable therein, prior to and/or afteradministering the composition.

Administration of the composition to a cell, tissue or organism mayfollow general protocols for the administration of cardiovasculartherapeutics, taking into account the toxicity, if any. It is expectedthat the treatment cycles would be repeated as necessary. In particularembodiments, it is contemplated that various additional agents may beapplied in any combination with the present invention.

a) Pharmacological Therapeutic Agents

Pharmacological therapeutic agents and methods of administration,dosages, etc. are well known to those of skill in the art (see forexample, the “Physicians Desk Reference”, Goodman & Gilman's “ThePharmacological Basis of Therapeutics”, “Remington's PharmaceuticalSciences”, and “The Merck Index, Eleventh Edition”, incorporated hereinby reference in relevant parts), and may be combined with the inventionin light of the disclosures herein. Some variation in dosage willnecessarily occur depending on the condition of the subject beingtreated. The person responsible for administration will, in any event,determine the appropriate dose for the individual subject, and suchindividual determinations are within the skill of those of ordinaryskill in the art.

Non-limiting examples of a pharmacological therapeutic agent that may beused in the present invention include an antihyperlipoproteinemic agent,an antiarteriosclerotic agent, an anti-cholesterol agent, ananti-inflammatory agent, an antithrombotic/fibrinolytic agent, a bloodcoagulant, an antiarrhythmic agent, an antihypertensive agent, or avasopressor. In certain aspects of the present invention,anti-cholesterolemic agents are used in combination with the compositionof the present invention. Anti-cholesterol agents include but are notlimited to HMG-CoA Reductase inhibitors, cholesterol absorptioninhibitors, bile acid sequestrants, nicotinic acid and derivativesthereof, fibric acid and derivatives thereof. HMG-CoA Reductaseinhibitors include statins, for example, but not limited to atorvastatincalcium (Lipitor®), cerivastatin sodium (Baycol®), fluvastatin sodium(Lescol®), lovastatin (Advicor®), pravastatin sodium (Pravachol®), andsimvastatin (Zocor®). Agents known to reduce the absorption of ingestedcholesterol include, for example, Zetia®. Bile acid sequestrantsinclude, but are not limited to cholestyramine, cholestipol andcolesevalam. Other anti-cholesterol agents include fibric acids andderivatives thereof (e.g., gemfibrozil, fenofibrate and clofibrate);nicotinic acids and derivatives thereof (e.g., nician, lovastatin) andagents that extend the release of nicotinic acid, for example niaspan.Anti-inflammatory agents include, but are not limited to non-steroidalanti-inflammatory agents (e.g., naproxen, ibuprofen, celeoxib) andsteroidal anti-inflammatory agents (e.g., glucocorticoids).

b) Surgical Therapeutic Agents

In certain aspects, a therapeutic agent may comprise a surgery of sometype, which includes, for example, preventative, diagnostic or staging,curative and palliative surgery. Surgery, and in particular a curativesurgery, may be used in conjunction with other therapies, such as thepresent invention and one or more other agents.

Such surgical therapeutic agents for cardiovascular diseases anddisorders are well known to those of skill in the art, and may comprise,but are not limited to, performing surgery on an organism, providing acardiovascular mechanical prostheses, angioplasty, coronary arteryreperfusion, catheter ablation, providing an implantable cardioverterdefibrillator to the subject, mechanical circulatory support or acombination thereof. Non-limiting examples of a mechanical circulatorysupport that may be used in the present invention comprise anintra-aortic balloon counterpulsation, left ventricular assist device orcombination thereof.

G. Autoimmune Disorders

According to the present invention, a subject that is suspected of anautoimmune disease or a subject suffering from an autoimmune disease istreated with the peptide composition of the present invention.

The pathogenesis of autoimmune disorders is mediated by the aberrantactivation, differentiation and trafficking of leukocytes in response totissue self-antigens. Autoimmune reactions can often have fatalconsequences. They can cause the destruction of vital tissue whichmanifests in the development of chronic autoimmune diseases. The drivingforces in the progression of these diseases are autoreactive immunecells. In most cases, these autoreactive cells are CD4+ T-cells whichsomehow escaped the self-tolerance control mechanisms of the immunesystem. In non-pathogenic situations, CD4+ T-cells act as T helpercells, which control or mediate the activation and differentiation ofother immune cells such as cytotoxic CD8+ T-cells, NK cells,granulocytes, macrophages and B-cells. However, in the situation ofchronic autoimmune diseases, CD4+ T-cells are no longer just mediators,but are rather key players of the autoimmune response. In particular,they are either directly or indirectly responsible for thecharacteristic tissue destruction that occurs in autoimmune diseases,which is triggered by the recognition of autoantigens. Theseautoantigens are derived from self-proteins of the attacked tissue andare presented to the autoreactive CD4+ T-cells by antigen-presentingcells (APCs), such as macrophages, dendritic cells or B-cells. Inaddition, aberrant T cell-dependent or T cell-independent antibodyproduction by activated B cells plays a critical role in initiating andamplifying tissue damage in autoimmune disorders.

Such autoimmune disorders that can be treated using the composition ofthe present invention include, but are not limited to, the following:Alopecia Areata, Ankylosing Spondylitis, Antiphospholipid Syndrome,aplastic anemia, myelodysplastic syndromes, paroxysmal nocturnalhemoglobulinemia, pure red cell aplasia, chronic neutropenias,amegakaryocytic thrombocytopenia, antiphospholipid syndromes, autoimmunethrombocytopenia, autoimmune hemolytic syndromes, antiphospholipidsyndromes, autoimmune gastritis, achlorhydria, Autoimmune Addison'sDisease, Autoimmune Diabetes, Autoimmune Hemolytic Anemia, AutoimmuneHepatitis, Autoimmune hypophysitis, Autoimmune orchiditis, autoimmuneovarian failure, Behcet's Disease, Bullous Pemphigoid, Cardiomyopathy,Celiac Sprue-Dermatitis, Cicatrical pemphigoid, Chronic Fatigue ImmuneDysfunction Syndrome (CFIDS), Chronic Inflammatory DemyelinatingPolyneuropathy, Interstitial cystitis, Churg-Strauss Syndrome,Cicatricial Pemphigoid, CREST Syndrome, Cold Agglutinin Disease, Crohn'sDisease, Dermatitis herpetiformis, Discoid Lupus, Drug-inducedautoimmune disorders, Endometriosis, Epidermolysis bullosa acquisita,Essential Mixed Cryoglobulinemia, Fibromyalgia-Fibromyositis,Glomerulonephritis, Good Pasture Syndrome, Graft Versus Host Disease,Graves' Disease, Guillain-Barré, Hashimoto's Thyroiditis, IdiopathicInflammatory Myopathies, Idiopathic Pulmonary Fibrosis, IdiopathicThrombocytopenia Purpura (ITP), IgA Nephropathy, Insulin DependentDiabetes, Juvenile Arthritis, Lichen Planus, Systemic LupusErythmatosis, Ménière's Disease, Metal-induced autoimmunity disorders,Mixed Connective Tissue Disease, Multiple Sclerosis, Myasthenia Gravis,Myocarditis, Myositis, Optic neuritis, Painless/postpartum thyroiditis,Peripheral nerve vasculitis, Pemphigus Foliaceus, Pemphigus Vulgaris,Pernicious Anemia, Polyarteritis Nodosa, Polychondritis, PolyglandularSyndromes, Polymyalgia Rheumatica, Polymyositis and Dermatomyositis,Postinfectious autoimmune disorders, Primary Agammaglobulinemia, PrimaryBiliary Cirrhosis, Psoriasis, Psoriatic Arthritis, Reactive Arthritis,Raynaud's Phenomenon, Reiter's Syndrome, Rheumatic Fever, RheumatoidArthritis, Sarcoidosis, Scleritis, Scleroderma, Sjögren's Syndrome,Stiff-Man Syndrome, Takayasu Arteritis, Temporal Arteritis/Giant-cellArteritis, Ulcerative Colitis, Uveitis, Vasculitis, Vitiligo, andWegener's Granulomatosis.

In certain embodiments, the composition of the present invention isadministered to a subject suffering from graft versus host disease(GVHD), organ transplant rejection, autoimmune hepatitis, primarybiliary cirrhosis, autoimmune cholangitis, primary sclerosingcholangitis, irritable bowel syndrome (IBS), multiple sclerosis (MS),chronic granulomatous disease, ankylosing spondylitis, scleroderma,polymyositis, (dermato)myositis, systemic vasculitis, systemic lupuserythematosus (SLE), Chrohn's disease, insulin-dependent diabetes(type 1) or ulcerative colitis.

1. Organ Transplant Rejection and GVHD

In accordance with the present invention, the composition provided inany of the above-described pharmaceutical carriers is administered as atherapeutic drug to a subject who has undergone organ transplant(kidney, heart, lung, liver, pancreas, bone marrow, peripheral stemcells) and/or as a prophylactic drug to organ and/or tissue transplant,for example bone marrow (BM) or peripheral stem cell donor cells, toprevent the development of GVHD in recipients and/or to prevent orattenuate transplant rejection in the recipients. One of skill in theart can determine the patients who would potentially benefit from atherapeutic agent that would reduce chronic allograft rejection andtoxicity associated with standard therapy, or the development of GVHD.

A further embodiment is treating, preventing or reducing the risk ofdeveloping graft-versus-host-disease by administering a lactoferrincomposition to the donor organ or donor tissue prior to transplantationinto the recipient. It is envisioned that administering the compositionto the donor tissue/organ will attenuate the immune cells in thedonor/organ and prevent the development of the immune response that ismounted against the recipient's tissue, thus preventing or attenuatingGVHD. In further embodiments, the recipient and the donor organ/tissuecan be treated with the composition of the present invention.

Additional embodiments of the present invention include a method oftreating, preventing or attenuating the severity of tissue or organtransplant rejection in a recipient comprising the step of administeringto the donor a lactoferrin composition in an amount sufficient toattenuate the tissue or organ transplant rejection in the recipient. Itis envisioned that the composition reduces allogeneic immune responsesin the recipient. The composition can also be administered to both therecipient and the donor.

Another embodiment is a method of treating, preventing or attenuatingthe severity of xenograft tissue or xenograft organ transplant rejectionin a recipient comprising the step of administering to the xenograftdonor a composition in an amount sufficient to attenuate the tissue ororgan transplant rejection in the recipient. The composition can also beadministered to both the recipient and the xenograft donor.

In particular embodiments, progenitor cells or stem cells may beisolated from at least one organ, cell, tissue or organism. Stem cellscan be isolated from embryonic or nonembryonic donors. The tissues fromwhich the stem cells can be isolated include, for example, but are notlimited to the bone marrow, the spleen, the liver, peripheral blood,umbilical cord tissue, umbilical cord blood, adipose tissue or skin. Yetfurther, tissue grafts may be used in the present invention. The tissuemay be part or separated from an organism. In certain embodiments, atissue may comprise, but is not limited to, adipocytes, alveolar,ameloblasts, axon, basal cells, blood (e.g., lymphocytes), blood vessel,bone, bone marrow, peripheral stem cells, brain, breast, cartilage,cervix, colon, cornea, embryonic, endometrium, endothelial, epithelial,esophagus, facia, fibroblast, follicular, ganglion cells, glial cells,goblet cells, kidney, liver, lung, lymph node, muscle, neuron, ovaries,pancreas, peripheral blood, prostate, skin, skin, small intestine,spleen, stem cells, stomach, testes, or ascite tissue.

In specific embodiments, an organ is the graft. Organs are comprised oftissues having a special function. Exemplary organs that are used ingrafts in the present invention include, but are not limited to heart,kidney, pancreas, lung, or liver.

In a preferred embodiment of the present invention, the composition isadministered in an effective amount to decrease, reduce, inhibit orabrogate chronic allograft rejection and toxicity related to standardtherapy. The amount of lactoferrin in the composition may vary fromabout 1 ng to about 100 g, more preferably, 1 mg to about 20 g.Preferably, the composition that is orally administered contains therange of 0.1 g to 5 g of lactoferrin per day.

The improvement is any observable or measurable improvement. Thus, oneof skill in the art realizes that a treatment may improve the patient orsubject's condition, but may not be a complete cure of the disease. Incertain aspects, the composition is administered in an effective amountto decrease, reduce, inhibit or abrogate levels of an immune responseagainst a graft in the recipient. In further aspects, an improvement canconsist of any of the following, for example, increased function of thegraft, for example, increased urine output for kidney grafts ordecreased jaundice for liver grafts; reduction in inflammation;reduction in general discomfort of the recipient; an overall increasedtolerance for the graft. Thus, after administration of lactoferrinrelated peptides, if any of the above conditions improve, then theamount is considered to be an effective amount.

An improvement in GHVD is also any observable or measurable improvement.Thus, one of skill in the art realizes that a treatment may improve thepatient or subject's condition, but may not be a complete cure of thedisease. In certain aspects, the composition is administered in aneffective amount to decrease, reduce, inhibit or abrogate levels ofimmune response from the donor's cells, tissue and/or organ against thehost's tissues. GVHD can be acute or chronic or mild or severe.Improvements in acute symptoms include any of the following, forexample, decrease skin rash, decrease diarrhea, increase in liverfunction, a decrease in susceptibility to infection. Improvements inchronic symptoms include, but are not limited to decrease skin rash,decrease dermatitis, decrease hair loss, a decrease liver damage,decrease dry eyes and mouth, a decrease susceptibility to infections anddecrease lung and/or gastrointestinal disorders. Thus, afteradministration of lactoferrin related peptides, if any of the aboveconditions improve, then the amount is considered to be an effectiveamount.

Still yet, a further embodiment is a method of regulating a mucosalimmune response in the gastrointestinal tract or a systemic immuneresponse in a subject comprising the step of administering a lactoferrinrelated peptide composition to said subject the composition of thepresent invention. It is envisioned that composition stimulates MIP-3αand interleukin-18 in the gastrointestinal tract, which regulates immuneresponses. For example, interleukin-18 modulates both Th1/Th2 responses.It is known by those of skill in the art that IL-18 plays an importantrole in allogeneic stem cell transplantation. Pre-treatment ofallogeneic donors with IL-18 significantly improves survival and reducesclinical and pathological indices of acute GVHD in BMT recipients. Othercytokines may also be enhanced or repressed for example, but not limitedto IL-1, IL-2, IL-6, IL10, IL-12 and GM-CSF. It is also envisioned thatlactoferrin stimulates Th1/Th2 type-responses through the inductionand/or repression of Th1 and Th2 cytokines.

In order to increase the effectiveness of administration of thecomposition of the present invention, it is desirable to combine thesecompositions with standard therapy. For example, known immunosuppressantagents are used in combination with the composition of the presentinvention. Exemplary agents known to prevent organ rejection are T cellmodifiers such as cyclosporine (Neoral®, Sandimmune®), prednisone (NovoPrednisone®, Apo Prednisone®), azathioprine (Imuran®), tacrolimus orFK506 (Prograf®), mycophenolate mofetil (CellCept®), OKT3 (MuromorabCO3®, Orthoclone®), ATGAM® & Thymoglobulin® or serine-threoninephosphatase calcineurin (CN) inhibitors. In specific embodiments, thestandard or approved treatment of GVHD, which is high dosescorticosteroids, primarily high-dose methylprednisolone, is used incombination with the lactoferrin composition of the present invention.

In addition to immunosuppressant other anti-rejection and/or anti-GVHDtherapies can be used in combination with the lactoferrin composition ofthe present invention. For example, therapies for preconditioning andprophylaxis of GVHD include, but are not limited to total bodyirradiation, cytosine arabinoside, L-phenylalanine mustard,cyclophosphamide, etoposide, triethylene thiophosphoramide,antithymocyte globulin, bisulfan, tacrolimus, methylprednisolone,cyclosporin, or methotrexate. Experimental therapies for treatment ofGVHD include, but are not limited to cytokine inhibitors/antagonists(e.g., anti-TNFα antibody); IL-1 receptor antagonist; recombinant IL-1receptor; inhibitors of T-cell activation (e.g., tacrolimus);antimetabolites (e.g., mycophenolate mofetil); anti-CD3 antibody(muromonab, OKT3); anti-CD25 antibody; anti-IL2 receptor monoclonalantibody daclizumab; extracorporeal photopheresis using ex vivo8-methoxypsoralen; anti-thymocyte globulins (Thymoglobulin® or ATGAM®);ABX-CBL or CBL-1; or Visilizumab (Nuvion®).

The composition of the present invention may precede, be co-current withand/or follow the other agent(s) by intervals ranging from minutes toweeks. In embodiments where the composition of the present invention,and other agent(s) are applied separately to a cell, tissue or organism,one would generally ensure that a significant period of time did notexpire between the time of each delivery, such that the composition andagent(s) would still be able to exert an advantageously combined effecton the cell, tissue or organism.

Various combination regimens of the composition and one or more agentsare employed. One of skill in the art is aware that the composition ofthe present invention and agents can be administered in any order orcombination.

H. Endocrine/Metabolism Disorders

1. Diabetes Mellitus

In accordance with the present invention, the composition provided inany of the above-described pharmaceutical carriers is administered to asubject who has experienced or is at high risk of having diabetesmellitus. Thus, it is envisioned that the composition of the presentinvention may be administered to a subject to regulate diabetesmellitus. The composition modulates at least one symptom of diabetesmellitus, for example, decrease blood glucose or modulate blood insulinlevels.

Risk factors for type I diabetes include islet-cell antibodies and thoseof type 2 or gestational diabetes include inactivity, obesity, siblingswith diabetes, and history of diabetes during pregnancy. One of skill inthe art can determine the patients who would potentially benefit from atherapeutic agent that would reduce circulating levels of glucose.

In a preferred embodiment of the present invention, the composition isadministered in an effective amount to decrease, reduce, inhibit orabrogate high glucose, or to reduce total body weight, glycosylatedhemoglobin (HbA1c), or blood pressure or to modulate blood insulinlevels. In the case of a diabetic condition, successful reduction ofhyperglycemia by the lactoferrin composition may be manifested by thefasting plasma glucose level falling below 126 mg/dL, the 2-hour plasmaglucose level during an oral glucose tolerance test (OGTT) falling belowbelow 200 mg/dL, or if a random plasma glucose determination readingbelow 200 mg/dL in a symptomatic individual. In the case of apre-diabetic condition, a successful reduction of hyperglycemia by thelactoferrin composition may be manifested by the fasting plasma glucosefalling below 110 mg/dL and/or the 2-hour plasma glucose on the OGTTfalling below between 140 mg/dL. The amount of lactoferrin relatedpeptides in the composition may vary from about 0.1 μg to about 100 g,more preferably, from about 1 μg to about 20 g, or any rangetherebetween. In specific embodiments, the composition that isadministered contains the range of 0.1 g to 5 g of lactoferrin per day.

Glycohemoglobin (or glycosylated hemoglobin) is measured to monitor oraccurately record blood glucose levels, and it is not influenced byacute changes in blood glucose or by the interval since the last meal.Glycohemoglobin is formed when glucose reacts non-enzymatically with thehemoglobin A molecule and is composed of several fractions, the majorone being HbA1c. Total glycohemoglobin (HbA1) and HbA1c (expressed asthe percentage of total hemoglobin) vary in proportion to the averagelevel of glucose over the lifespan of the red blood cell (RBC), therebyproviding an index of glycemic control.

Further aspects of the invention include reducing blood glucose in apatient suffering from diabetes mellitus by administering to a subjectan effective amount of the composition such that the amount of thelactoferrin related peptides modulates blood glucose. The blood glucoseis monitored by the level of glycosylated hemoglobin (HbA1c).

Another aspect is a method of modulating blood insulin in a patientsuffering from diabetes mellitus by administering to a subject aneffective amount of the composition such that the amount modulates bloodinsulin. Modulating blood insulin includes reducing or maintaining bloodinsulin levels or increasing blood insulin levels. Modulating bloodinsulin levels in the present invention includes increasing bloodinsulin levels in insulin-dependent diabetes (Type I) or decreasing ormaintaining insulin levels in non-insulin-dependent diabetes (Type II).

In order to increase the effectiveness of administration of thecomposition of the present invention, it is desirable to combine thecompositions with an additional agent. For example, known diabetesagents are used in combination with the composition of the presentinvention. Exemplary agents known to treat high glucose or insulinsulfonylureas, biguanides, alpha-glucosidase, thiazolidinedione,meglitinide, and amino acid D-phenylalanine derivative. Otherantidiabetic agents may also include, a weight loss regimen and/or adiet alteration.

The composition of the present invention may precede, be co-current withand/or follow the other agent(s) by intervals ranging from minutes toweeks. In embodiments where the composition of the present invention,and other agent(s) are applied separately to a cell, tissue or organism,one would generally ensure that a significant period of time did notexpire between the time of each delivery, such that the composition andagent(s) would still be able to exert an advantageously combined effecton the cell, tissue or organism.

Various combination regimens of the composition and one or more agentsare employed. One of skill in the art is aware that the composition ofthe present invention and agents can be administered in any order orcombination.

2. Treatment of Fragile Bone Diseases

In accordance with the present invention, the peptide compositionprovided in any of the above-described pharmaceutical carriers isadministered to a subject suspected of or having a fragile bonecondition.

The present invention is designed for the treatment of any type offragile bone condition or any disorder relating to bone loss includingbut not limited to osteoporosis, age-associated osteoporosis,postmenopausal osteoporosis, osteitis deformans (Paget's disease),osteogenesis imperfecta (brittle bones), and osteopetrosis.

Bone loss disorders arise from an imbalance in the formation of newhealthy bone and the resorption of old bone, skewed toward a net loss ofbone tissue. This bone loss includes a decrease in both mineral contentand protein matrix components of the bone, and leads to an increasedfracture rate of, predominantly, femoral bones and bones in the forearmand vertebrae. These fractures, in turn, lead to an increase in generalmorbidity, a marked loss of stature and mobility, and, in many cases, anincrease in mortality resulting from complications.

Bone loss occurs in a wide range of subjects, including postmenopausalwomen, patients who have undergone hysterectomy, patients who areundergoing or have undergone long-term administration ofcorticosteroids, patients suffering from Cushing's syndrome, andpatients having gonadal dysgensis.

Unchecked, bone loss can lead to osteoporosis, a major debilitatingdisease whose prominent feature is the loss of bone mass (decreaseddensity and enlargement of bone spaces) without a reduction in bonevolume, producing porosity and fragility.

Osteoporosis is common in the elderly of both sexes but is morepronounced in postmenopausal women. Osteoporosis may occur as a primarydisorder or as a secondary complication of several diseases. It isproposed that genetic factors determine the size of the bone massachieved in young adulthood. With aging, the increased osteoclasticfunction and the slowing of osteoblastic activity induced by endocrineinfluences, particularly decreased estrogen levels, result in a netnegative balance in the continued turnover of bone. Osteoporosis causesbone pain owing to microfractures; results in loss in height andstability of the vertebral column; and predisposes to fractures offemoral necks, wrists, and vertebrae. The condition remains asymptomaticuntil skeletal fragility is well advanced.

Paget's disease is currently considered to be a slow paramyxoviralinfection of osteoblasts and then osteoclasts. The condition is dividedinto an initial osteolytic stage, followed by a mixedosteolytic-osteoblastic stage, evolving ultimately into burn-outquiescent osteosclerotic stage. Because new bone formation in activedisease is disordered and poorly mineralized, it is soft and porous,lacks structural stability, and is vulnerable to fracture or deformationunder stress. Patients may demonstrate fractures, nerve compression,osteoarthritis, and skeletal deformities.

Osteogenesis imperfecta or brittle bones refers to a group of closelyrelated genetic disorders caused by qualitative or quantitative abnormalsynthesis of type I collagen, constituting about 90% of the matrix ofbone. Syndromes range from one variant (type II) that is uniformly fatalin the perinatal period (from multiple bone fractures) to other variantsmarked by increased predisposition to fracture but compatible withsurvival. Morphologically the basic change in all is osteopenia or toolittle bone, with marked thinning of the cortices and rarefication ofthe trabeculae.

Osteopetrosis refers to a group of rare hereditary diseasescharacterized by overgrowth and sclerosis of bone, with markedthickening of the cortex and narrowing or filling of the medullarycavity impairing hematopoiesis. Despite too much bone, it is brittle andfractures easily. The autosomal recessive form is evident from birth,with anemia, neutropenia, infections and eventual death. The autosomaldominant form is benign but predisposes to fractures. Common to allforms is a hereditary defect in osteoclast function resulting in reducedbone resorption and enhanced net bone overgrowth.

In a preferred embodiment of the present invention, lactoferrin relatedpeptides are administered in an effective amount to stabilize or reducebone fragility, to strengthen the bone, inducing the growth of healthybone, modulate calcium levels, and modulate mineral accumulation in theskeleton. The amount may vary from about 1 μg to about 100 g oflactoferrin related peptides. Preferably, the amount that isadministered is in the range of 10 mg to 25 g. Most preferably, theamount that is administered is in the range of 10 μg to 5 g.

It is often desirable to deliver the composition of the presentinvention to the subject over prolonged periods of time, for example,for periods of one week to one year from a single administration.Various slow release, depot or implant dosage forms may be utilized. Forexample, a dosage form a may contain a pharmaceutically acceptablenon-toxic salt of the compounds which has a low degree of solubility inbody fluids, for example, (a) an acid addition salt with a polybasicacid such as phosphoric acid, sulfuric acid, citric acid, tartaric acid,tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalenemono- or di-sulfonic acids, polygalacturonic acid, and the like; (b) asalt with a polyvalent metal cation such as zinc, calcium, bismuth,barium, magnesium, aluminum, copper, cobalt, nickel, cadmium and thelike, or with an organic cation formed from e.g.,N,N′-dibenzyl-ethylenediamine or ethylenediamine; or (c) combinations of(a) and (b) e.g. a zinc tannate salt. Additionally, the compounds of thepresent invention or, preferably, a relatively insoluble salt such asthose just described, may be formulated in a gel, for example, analuminum monostearate gel with, e.g. sesame oil, suitable for injection.Particularly preferred salts are zinc salts, zinc tannate salts, pamoatesalts, and the like. Another type of slow release depot formulation forinjection would contain the compound or salt dispersed for encapsulationin a slow degrading, non-toxic, non-antigenic polymer such as apolylactic acid/polyglycolic acid polymer for example as described inU.S. Pat. No. 3,773,919. The compounds or, preferably, relativelyinsoluble salts such as those described above may also be formulated incholesterol matrix silastic pellets, particularly for use in animals.Additional slow release, depot or implant formulations, e.g. liposomes,are well known in the literature. See, for example, “Sustained andControlled Release Drug Delivery Systems”, J. R. Robinson ed., MarcelDekker, Inc., N.Y., 1978.

Still yet, a further embodiment is a method of promoting the stimulationor inhibition of cytokines, growth factors or mediators of bone growthor resorption. Such growth factors include, but not limited toinsulin-like growth factor-1 (IGF-1), transforming growth factor-beta(TGF-b), and growth hormone releasing factor (GRF). Mediators of bonegrowth or resorption include, but are not limited to parathyroid hormone(PTH), calcitonin or 1,25-dihydroxycholecalciferol (1,25(OH)2D3).

Still yet, a further embodiment is a method of activating or inhibitingcells involved in bone remodeling including but not limited toosteoblasts, osteoclasts, chondrocytes, endothelial cells, andfibroblasts.

In order to increase the effectiveness of the composition of the presentinvention, it may be desirable to combine the composition of the presentinvention with other agents effective in the treatment of fragile bonediseases including but not limited to calcium and cholecalciferol(vitamin D) supplements, estrogen replacement therapy (ERT), raloxifene,calcitonin, bisphosphonates such as alendronate and cyclical etidronate,statins, recombinant human parathyroid hormone (rhPTH), insulin-likegrowth factor-1 (IGF-1), and transforming growth factor-beta (TGF-1).This process may involve administering the lactoferrin composition ofthe present invention and the agent(s) or multiple factor(s) at the sametime. This may be achieved by administering a single composition orpharmacological formulation that includes both agents, or byadministering two distinct compositions or formulations, at the sametime, or at times close enough so as to result in an overlap of thiseffect, wherein one composition includes the human lactoferrincomposition and the other includes the second agent(s).

Alternatively, the composition of the present invention may precede orfollow the other bone healing agent treatment by intervals ranging fromminutes to weeks. In embodiments where the other bone healing agent andthe inventive composition are administered or applied separately, onewould generally ensure that a significant period of time did not expirebetween the time of each delivery, such that the agent and thecomposition would still be able to exert an advantageously combinedeffect on the condition.

I. Immunosuppressed Subjects

In order to carry out methods of the present invention, the compositionof the present invention is administered to an immunosuppressedindividual or an individual whose immune system is expected to besuppressed.

As noted above, the subject method is suited for preventing infectionand/or inflammation in immunosuppressed individuals or individuals whoseimmune systems are expected to be repressed. In one aspect, theimmunosuppressed subject exhibits neutropenia, or suffers from AIDS orany form of cancer including lymphoma and myeloma. In yet anotheraspect, the subject undergoes or is expected to undergo a therapyselected from the group consisting of chemotherapy, radiotherapy, andautologous peripheral stem cell transplant.

An effective amount of the lactoferrin composition depends on theseverity and/or course of immunosuppression, the patient's clinicalhistory and response, and the discretion of the attending physician. Thecomposition is suitably administered to the patient at one time or overa series of treatments. The initial candidate dosage may be administeredto a patient. The proper dosage and treatment regimen can be establishedby monitoring the progress of therapy using conventional techniquesknown to the people skilled of the art.

The amount of active ingredients that may be combined with the carriermaterials to produce a single dosage form will vary depending upon thesubject treated and the particular mode of administration. It will beunderstood, however, that the specific dose level for any particularpatient will depend upon a variety of factors, including the activity ofthe specific composition employed, the age, body weight, general health,sex, diet, time of administration, route of administration, and rate ofexcretion, drug combination and the severity of the particular diseaseundergoing therapy, and can be determined by those skilled in the art.

In order to achieve effective prophylaxis, preferably, the lactoferrincomposition is administered prior to the inception neutropenia, morespecifically Grade 3 or higher neutropenia and continued on the sameschedule daily for the entire period during which the patient is at ahigh risk of neutropenic fever, infection or mucositis.

It is envisioned that the immune response is enhanced by lactoferrinstimulating cytokines and/or chemokines. Exemplary cytokines includeinterleukin-18 and GM-CSF in the gastrointestinal tract, which are knownto enhance immune cells or stimulate production of immune cells.

Certain treatment regimens include administering the composition inwhich the concentration of lactoferrin related peptides that is providedis in the range of 1 g to about 100 g/ml and the composition is asolution that is swished in the mouths of the subjects for a few minutes(e.g., about 4 minutes) and then swallowed. This process can be repeatedmultiple times a day, for example twice or more each day, for a total ofthree doses per 24 hour period. The doses are taken immediately aftermeals (one each after breakfast, lunch and dinner), or at similarlyspaced intervals throughout the day. Subjects are asked not to eat ordrink anything for a least one hour after swallowing the dose. Thetreatment may be terminated in the event of significant drug relatedadverse effects observed during one of the interim safety analyses.

The multiple administration of lactoferrin related peptides each day canmaximize the exposure of the oral cavity and gut to the drug.Preferably, patients an effective amount of the compositionprophylactically for 21-28 days starting from the commencement ofchemotherapy. Daily dosing continues through the neutropenic phase untilthe earliest of the following: a) Absolute Neutrophil Count (ANC) is≧600 cells/μL for three consecutive days, and mucositis (if present) hasimproved to Grade 2 or better for three consecutive days; or b) patientis discharged from the hospital or outpatient transplant setting. Alast-day-of-therapy evaluation is performed within 24 hours after thelast dose. The patients are also required to return for anend-of-treatment evaluation, to be performed 3-7 days after the lastdose.

The treatment regimen in the present invention can prevent or attenuateinfections in immunosuppressed patients, for example, patientsundergoing aggressive chemotherapy or radiotherapy. The treatmentreduces incidence, duration, and severity, and prolongs theinfection-free interval of neutropenic infection (neutropenic fever).For the purpose of this invention, fever is considered as an oraltemperature of at least 38.0° C. on at least two occasions within 24hours, or a single oral temperature of at least 38.3° C. Neutropenicfever is defined as fever that begins when the patient is neutropenic(ANC<500). The end of neutropenic fever is defined as the point at whichthe patient begins a period of 48 hours without fever (as definedabove), whether or not the patient is still neutropenic. Incidence ofneutropenic fever is calculated as the number of patients experiencingone or more episodes of neutropenic fever. Onset of neutropenic fever isdetermined in two ways: (1) days between the transfusion and the firstepisode of neutropenic fever and (2) days between a neutrophil count<1000/μL and the first episode of neutropenic fever. Duration ofneutropenic fever is defined as the number of days between the onset andthe end of neutropenic fever. Severity of neutropenic fever isdetermined by the average temperature of the patient during the periodof neutropenic fever. Infection-free interval is the period between theend of an infective fever and the onset of the next infective fever.

The present treatment also reduces the incidence, severity, and durationof oral and GI mucositis by improving all clinical criteria used formucositis evaluation (pain, salivation, appetite, and oralinflammation). The incidence, duration, and severity of oral mucositisare assessed for each patient. Oral mucositis is graded using the OralMucositis Assessment Scale (OMAS).

Prophylactic and concomitant use of antibiotics may be used during thetreatment according to patient's conditions and usual standard of carein the medical profession. Use of hematopoietic growth factors is notrecommended routinely, but G-CSF may be used as part of the standard ofclinical carte at the discretion of the doctors. All medicationsnecessary for the patient's well being may also be administered.

The responses of the patients in the course of treatment are closelymonitored by evaluating various response parameters. The treatmentprotocol may be adjusted accordingly depending on the improvement of thepatient's physical conditions.

J. Gastrointestinal Disorders

In accordance with the present invention, the peptide compositionprovided in any of the above-described pharmaceutical carriers isadministered to a subject suspected of or having a gastrointestinaldisorder.

Gastrointestinal disorders include, but are not limited to, heartburn,bloating, postoperative ileus, abdominal pain and discomfort, earlysatiety, epigastric pain, nausea, vomiting, burbulence, regurgitation,intestinal pseudoobstruction, anal incontinence, gastroesophageal refluxdisease, irritable bowel syndrome, ulcerative colitis, Crohn's disease,menstrual cramps, pancreatitis, spastic and interstitial cystitis andulcers and the visceral pain associated therewith.

In certain embodiments, the peptide composition is administered to asubject suffering from Crohn's disease, colitis, necrotizingenterocolitis, endometriosis, irritable bowel syndrome, pancreatitis,periodontal disease, and ulcerative colitis.

K. Neurological Conditions

In accordance with the present invention, the peptide compositionprovided in any of the above-described pharmaceutical carriers isadministered to a subject suspected of or having a neurologicaldisorder.

It is envisioned that the composition of the present invention whenadministered to a subject suffering from a neurological disorder,stimulates the immune system thereby alleviating the neurologicalconditions. Thus, the composition of the present invention can be usefulwith regard to the prevention, treatment, or amelioration ofneurological, psychiatric, psychological, conscious state, behavioral,mood, and thought activity (unless otherwise indicated these will becollectively referred to herein as “neurological activity” whichincludes “psychological activity” or “psychiatric activity”). Whenreferring to a pathological or undesirable condition associated with theactivity, reference may be made to a neurological disorder whichincludes “psychiatric disorder” or “psychological disorder” instead ofneurological activity or psychiatric or psychological activity. Althoughthe activity to be modulated usually manifests itself in the form of adisorder such as a attention or cognitive disorders (e.g., AutisticSpectrum Disorders); mood disorder (e.g., major depressive disorder,bipolar disorder, and dysthymic disorder) or an anxiety disorder (e.g.,panic disorder, posttraumatic stress disorder, obsessive-compulsivedisorder and phobic disorder); neurodegenerative diseases (e.g.,multiple sclerosis, Alzheimer's disease, amyotrophic lateral sclerosis(ALS), Parkinson's disease, Huntington's Disease, Guillain-Barresyndrome, myasthenia gravis, sleep disorders, and chronic idiopathicdemyelinating disease (CID)), one skilled in the art appreciates thatthe invention may also find application in conjunction with enhancing ordiminishing any neurological or psychiatric function, not just anabnormality or disorder.

In certain embodiments, the lactoferrin related peptide composition isadministered to a subject suffering from multiple sclerosis, Alzheimer'sdisease, Parkinson's disease, muscular dystrophy, sleep or depression.

Neurological are diagnosed based upon the accumulation of physical,chemical, and historical behavioral data on each patient. One of skillin the art is able to perform the appropriate examinations to accumulatesuch data. One type of examination can include neurologicalexaminations, which can include mental status evaluations, which canfurther include a psychiatric assessment. Other types of examinationscan include, but are not limited to, motor examination, cranial nerveexamination, and neuropsychological tests (e.g., Minnesota MultiphasicPersonality Inventory, Beck Depression Inventory, or Hamilton RatingScale for Depression). Thus, any improvement is based upon animprovement in any of the examinations that are used to diagnose aneurological disorder.

In addition to the above examinations, imaging techniques can be used todetermine normal and abnormal brain function that can result indisorders. Functional brain imaging allows for localization of specificnormal and abnormal functioning of the nervous system. This includeselectrical methods such as electroencephalography (EEG),magnetoencephalography (MEG), single photon emission computed tomography(SPECT), (SPECT), as well as metabolic and blood flow studies such asfunctional magnetic resonance imaging (fMRI), and positron emissiontomography (PET) which can be utilized to localize brain function anddysfunction.

In order to increase the effectiveness of administration of thecomposition of the present invention, it is desirable to combine thecompositions with an additional agent. For example, known agents thatare known to be stimulatory or inhibitory are used in combination withthe composition of the present invention to treat a variety ofneurological conditions.

Herein, stimulating drugs comprise medications, anesthetic agents,synthetic or natural peptides or hormones, neurotransmitters, cytokinesand other intracellular and intercellular chemical signals andmessengers, and the like. In addition, certain neurotransmitters,hormones, and other drugs are excitatory for some tissues, yet areinhibitory to other tissues. Therefore, where, herein, a drug isreferred to as an “excitatory” drug, this means that the drug is actingin an excitatory manner, although it may act in an inhibitory manner inother circumstances and/or locations. Similarly, where an “inhibitory”drug is mentioned, this drug is acting in an inhibitory manner, althoughin other circumstances and/or locations, it may be an “excitatory” drug.

Similarly, excitatory neurotransmitter agonists (e.g., norepinephrine,epinephrine, glutamate, acetylcholine, serotonin, dopamine), agoniststhereof, and agents that act to increase levels of an excitatoryneurotransmitter(s) (e.g., edrophonium; Mestinon; trazodone; SSRIs(e.g., flouxetine, paroxetine, sertraline, citalopram and fluvoxamine);tricyclic antidepressants (e.g., imipramine, amitriptyline, doxepin,desipramine, trimipramine and nortriptyline), monoamine oxidaseinhibitors (e.g., phenelzine, tranylcypromine, isocarboxasid)),generally have an excitatory effect on neural tissue, while inhibitoryneurotransmitters (e.g., dopamine, glycine, and gamma-aminobutyric acid(GABA)), agonists thereof, and agents that act to increase levels of aninhibitory neurotransmitter(s) generally have an inhibitory effect.(Dopamine acts as an excitatory neurotransmitter in some locations andcircumstances, and as an inhibitory neurotransmitter in other locationsand circumstances.) However, antagonists of inhibitory neurotransmitters(e.g., bicuculline) and agents that act to decrease levels of aninhibitory neurotransmitter(s) have been demonstrated to excite neuraltissue, leading to increased neural activity. Similarly, excitatoryneurotransmitter antagonists (e.g., prazosin, and metoprolol) and agentsthat decrease levels of excitatory neurotransmitters may inhibit neuralactivity. Yet further, lithium salts and anesthetics (e.g., lidocane)may also be used in combination with the present invention.

In specific embodiments, antidepressants are administered in combinationwith the present invention to treat an affective disorder, such asdepression. Such antidepressants include, but are not limited tomonoamine oxidase inhibitors, 5-HT1A agonists, selective 5-HT uptakeinhibitors, imipramine or clomipramine.

The composition of the present invention may precede, be co-current withand/or follow the other agent(s) by intervals ranging from minutes toweeks. In embodiments where the composition of the present invention,and other agent(s) are applied separately to a cell, tissue or organism,one would generally ensure that a significant period of time did notexpire between the time of each delivery, such that the composition andagent(s) would still be able to exert an advantageously combined effecton the cell, tissue or organism.

Various combination regimens of the composition and one or more agentsare employed. One of skill in the art is aware that the composition ofthe present invention and agents can be administered in any order orcombination.

L. Hematological Disorders

In accordance with the present invention, the peptide compositionprovided in any of the above-described pharmaceutical carriers isadministered to a subject suspected of or having a hematologicaldisorder. Such disorders can include, but are not limited to anemia,sickle cell anemia or cachexia.

M. Ocular Disorders

In accordance with the present invention, the peptide compositionprovided in any of the above-described pharmaceutical carriers isadministered to a subject suspected of or having an ocular disorder.Such disorders include, but are not limited to conjunctivitis, dry eyedisease, glaucoma, allergic eye disease, uveitis or ocular infection.

Dry eye disease or keratoconjunctivitis sicca affects about 4 millionpeople in the U.S. of which about 1.5 million have moderate to severedisease. Globally, 60 million people use artificial tears (Damato et al.1984, Hikichi et al. 1995, Bjerrum et al. 1997, Schein et al. 1997).Despite the high levels of incidence, there is currently no highlyeffective treatment. Development of therapeutic treatments has beenhampered by the lack of knowledge regarding the etiology of thecondition to its complexity. However, it has now been suggested thatinflammation affecting the lacrimal gland and ocular surface underliesthe pathophysiological process (Pflugfelder et al. 1986, Raphael et al.1988, Pepose et al. 1990, Kroemer et al. 1991, Jones et al. 1994,Pflugfelder et al. 1999, Turner et al. 2000). Thus, it is contemplatedthat the peptide composition of the present invention can be used todecrease the inflammatory response thereby treating the ocular diseaseor disorder.

In certain embodiments, the lactoferrin compositions are preferablyformulated as topical ophthalmic suspensions or solutions, with a pH ofabout 4 to 8. The establishment of a specific dosage regimen for eachindividual is left to the discretion of the clinicians. The amount oflactoferrin will normally be contained in these formulations in anamount 0.01% to 5% by weight, but preferably in an amount of 0.05% to 2%and most preferably in an amount 0.1 to 1.0% by weight. The dosage formmay be a solution, suspension microemulsion. Thus, for topicalpresentation 1 to 2 drops of these formulations would be delivered tothe surface of the eye 1 to 4 times per day according to the discretionof a skilled clinician.

V. Examples

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Anti-Microbial Effects of said Peptides

The novel peptides described in this application have shown substantialanti-microbial activity. Peptide MICs were determined by the brothmicrodilution method recommended by the NCCLS (M7-A5). In each medium,the concentrations of peptide were serial two-fold dilutions rangingfrom 8192 to 8 μg/mL. Mueller-Hinton broth and Peptone broth were usedthroughout, using the following media: 1) 5rCAMHB, 2) CAMHB supplementedwith 3% lysed horse blood, 3) Haemophilus test medium, 4) peptone brothat pH 7, 5) peptone broth at pH 7.5, 6) peptone broth supplemented with25 μg/mL calcium and 12.5 μg/mL magnesium, 7) peptone broth supplementedwith 1.5 g/L soluble starch. On each day of testing, colony counts wereperformed on the broth from the growth control well immediately afterinoculation from two randomly selected isolates to assure appropriateinoculum density. A total of 742 clinical bacterial isolatesrepresenting 35 species were tested. TABLE 2 MIC Geomeans for Said NovelPeptides Sequence Position Peptide 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1516 17 18 19 20 LPF-21 R R R R R R S V Q W C A V S Q P E A T K LPF-22 R RR R R R V Q W C A V S Q P E A T K C LPF-23 R R R R R R S V Q W C A V S QP E A T K LPF-24 R R R R R R S V Q W Q A V S Q P I A T E LPF-25 R R R RR R S V Q W A A V S Q P I A T E LPF-31 R R R R R R S V Q W Q A V S Q P EA T K LPF-32 R R R R R R S V Q W Q A V S Q P G A T K LPF-33 R R R R R RS V Q W Q A V S Q P I A T K LPF-34 R R R R R R S V Q W Q A V S Q P Q A TG LPF-35 R R R R R R S V Q W Q A V S Q P I A T I LPF-36 R R R R R R S VQ W A A V S Q P I A T K Sequence Position Peptide 21 22 23 24 25 26 2728 29 30 31 32 33 34 MIC GeoMean LPF-21 C F Q W Q R N M R K R R R 84LPF-22 F Q W Q R N M R K R R R R 91 LPF-23 C F Q W Q R N M R K R R R R84 LPF-24 Q F Q W Q R N M R K R R R 43/51 LPF-25 A F Q W Q R N M R K R RR 66 LPF-31 Q F Q W Q R N M R K R R R 41 LPF-32 Q F Q W Q R N M R K R RR 25 LPF-33 Q F Q W Q R N M R K R R R 28 LPF-34 Q F Q W Q R N M R K R RR 32 LPF-35 Q F Q W Q R N M R K R R R 15 LPF-36 A F Q W Q R N M R K R RR 24

Example 2 Stimulation of Immune Response by LPF-35

Balb/c naïve mice were treated orally with LPF-35 (5 mg/kg) or placeboonce a day for 3 days. One day later (day 4), mice were sacrificed andspleens & whole blood (PBMC) were collected. Spleen: NK cells wereseparated and counted. Cells were tested in vitro for NK-activity(FIG. 1) against YAC targets using an LDH-releasing test. PBMC: cellswere analyzed by FACS for the expression of the following T-cellmarkers: CD3/CD4, CD3/CD8, CD4/CD69 and CD8/CD69. T-cell activity isshown in FIG. 2.

Example 3 LPF-35 in a Mouse Paw Edema Model of Inflammation

Using the following experimental arrangement it was determined thatLPF-35 acts as a systemic anti-inflammatory agent after oraladministration. Test substance LPF-35 at 5 mg/kg and reference agentAspirin at 100 mg/kg were administered orally once daily for 5consecutive days to groups of 8 ICR (4 male and 4 female) derived miceweighing 22±2 gms. One hour after 5th dose, right hind paw was injectedwith carrageenan (0.05 ml of 1% suspension intraplantar). The hind pawedema was measured 4 hours later. Convention: reduction of hind pawedema by 30 percent or more indicates significant acuteanti-inflammatory activity. Each animal was fasted overnight beforeCarrageenan challenge.

The Table 3 below shows the average (n=8) paw swelling volume (mL/100).TABLE 3 Paw Volume (× 0.01 ml) % Treatment Route Dose RP LP Diff.Inhibition. Vehicle PO  5 ml/kg × 5 19.63 12.25 7.38 — (PBS Buffer)LPF-35 PO  5 mg/kg × 5 18.75 12.63 6.13 17 Aspirin PO 100 mg/kg × 517.38 12.38 5.00 32LPF35 vs vehicle, unpaired t test, one-tailed p = 0.0101LPF35 vs. Aspirin, unpaired t test, one-tailed p = 0.0233

Example 4 Effect of LPF-35 in Lipopolysaccharide-Induced Septic Shock inMice

An LPS-induced model of endotoxemia was used to demonstrate that LPF-35is effective in reducing mortality in a mouse model of sepsis. Groups of8-10 C57BL/6J mice 18±1 g were used. Animals received 20 ng/mouse dosesof E. coli Lipopolysaccharide (LPS) (IV) immediately after pre-treatmentwith D(+)-Galactosamine (20 mg/mouse IV). Animals were treated by anoral administration of LPF-35 at 1, 6 and 12 hours after challenge withLPS. Mortality was recorded every 12 hours over a 3-day period. In thismodel, reduction of mortality by 50% or more relative to thevehicle-treated group indicates significant protection. In thisexperiment doses of LPF-35 ranging between 5-100 μg were tested. TABLE 4Treatment Route & dose N of animals used % Survival LPF-35  5 μg × 3,p.o. 8 25 LPF-35  25 μg × 3, p.o. 8 25 LPF-35 100 μg × 3, p.o. 8 50vehicle  0 μg × 3, p.o. 8 0

Example 5 Effect of LPF-35 in a Mouse Model of Septic Shock

In a second experiment with LPF-35 in a mouse model of sepsis thehighest effective dose of LPF-35 was shown to be more effective than apositive control, recombinant human lactoferrin (rhLF). Groups of 8-16C57BL/6J mice 18±1 g were used. Animals received 20 ng/mouse doses of E.coli Lipopolysaccharide (LPS) (IV) immediately after pre-treatment withD(+)-Galactosamine (20 mg/mouse IV). Animals were treated by an oraladministration of LPF-35, rhLF or vehicle control at 1, 6 and 12 hoursafter challenge with LPS. Mortality was recorded every 12 hours over a3-day period. Results of Experiments with LPF-35 and rhLF in a Model ofSepsis are shown in FIG. 3.

Example 6 LPF-35 in a Rabbit Model of Dry Eye Disease

Experimental evidence suggests that peptide LPF-35 is effective in ananimal model of dry eye disease. Rabbits were dosed in the eyes with 50uL of isotonic saline (Vehicle n=6) or LPF-35 peptide (n=3) and the eyeswere held open for 2 hr except for the Background (untreated) group(n=3). After sacrifice, the corneas were stained with methylene blue,the stain extracted with acetone and sodium sulfate and analyzed with aspectrophotometer at absorbance 660 nm (Abs 660 nm) as a measure of eyedamage. Results of experiments with LPF-35 in a Model of Dry Eye Diseaseare shown in FIG. 4.

Example 7 LPF-32 and LPF-35 in a Sheep Model of Allergic Asthma

Peptides LPF-32 and LPF-35 were both tested in an established sheepmodel of allergic asthma. In both cases these peptides demonstrated theability to substantially inhibit late phase inflammation and airwayhyperresponsiveness. Allergic sheep previously characterized as dualresponders (displaying early and late phases of bronchoconstriction),were administered peptide followed by inhalation challenge with Ascarissuum antigen.

Baseline dose response curves to aerosolized carbachol were obtained inall sheep 1-3 days before the start of dosing. Sheep were dosed withpeptide or placebo by nebulization or oral gavage in the inhaled andoral experiments respectively. The sensitized sheep were then challengedwith Ascaris suum antigen to produce early and late airway responses, asmeasured by mean lung resistance (pulmonary flow resistance, RL). Meanlung resistance was measured using tracheal and pleural pressurecatheters connected to a differential pressure transducer. On thechallenge day measurements of RL were obtained before the antigenchallenge and then repeated 30 min after rhLF treatment, after which thesheep were challenged with Ascaris suum antigen. Measurements of RL wereobtained immediately after challenge, and hourly from 1-6 h afterchallenge and on the half-hour from 6½-8 h after challenge. Measurementsof RL were obtained 24 h after challenge followed by the 24 h postchallenge dose response curve.

To assess airway responsiveness, cumulative dose response curves tocarbachol were obtained, as measured by specific lung resistance, or SRL(SRL=RL * thoracic gas volume). The sheep inhaled a saline solution,then 10 consecutive breaths of increasing carbachol concentrations up to4% wt/vol. The provocation test was discontinued when SRL increased 400%from the post saline value, or after the highest carbachol concentrationhad been administered. Airway hypersensitivity (AH) was determined bycalculating the cumulative carbachol dose that increased SRL by 400%over the post-saline value by interpolation from the dose responsecurve. Actively dosed animals were compared to placebo treated animalsor their own historical controls. Results of Experiments with InhaledLPF-32/35 in a Sheep Model of Asthma are shown in FIG. 5. Results ofExperiments with Oral LPF-35 in a Sheep Model of Asthma are shown inFIG. 6.

Example 8 LPF-35 in Wound Healing

Experiments have shown that LPF-35 is effective in accelerating the rateand incidence of wound healing. Groups of 7 ICR male mice wereanesthetized, the shoulder and back region of each animal was shaved,and a sharp punch (ID 12 mm) was used to remove the skin includingpanniculus carnosus and adherent tissues. The wound area, traced ontoclear plastic sheets on days 3, 5, 7, 9 and 11 or 12, was quantitatedwith an Image Analyzer. An LPF-35 solution and placebo (PBS) wereapplied topically at 0.02 ml per wound immediately following injury andonce daily thereafter for a total of 11 consecutive days. The unpairedStudent's t test was applied for comparison between treated and vehiclegroup. Differences were considered statistical significant at P<0.05. Inaddition, the incidence of animals reaching 75% wound closure wasassessed and differences compared using Fisher's exact test. Differenceswere considered of statistical significance at p<0.05 levels. Results ofexperiments with Oral LPF-35 in a Mouse Model of Wound Healing are shownin FIG. 7.

Example 9 LPF-35 in a Mouse Model of Diabetes

In this experiment LPF-35 was tested in a mouse model of diabetes. C57BLmice (n=4) were used to test serum glucose and insulin levels in treatedmice versus controls. Mice were administered oral LPF-3 once daily for 3days. Baseline glucose and insulin levels were determined and comparedto levels 90 minutes after the last dose. The percentage change wasrecorded. LPF-35 produced significant declines in both serum glucose andinsulin relative to placebo. Results of Experiments with Oral LPF-35 ina Mouse Model of Diabetes are shown in FIG. 8.

Example 10 LPF-35 Stimulates MIP3-α In Vitro

In this experiment it was demonstrated, in vitro, that LPF-35 stimulatesthe expression of MIP3-α in Hepatocytes. MIP3-α has been associated withthe stimulation of innate immune responses. Using a 96-well format, weexposed tissue samples to various concentrations of placebo or testcompound (LPF-35 at 0.04 and 0.2 mg/mL). Supernatant was collected (in75 microL aliquots) from cells at 2 h and 4 h after the administrationof test compounds and was tested for concentration of MIP3-α by ELISA.LPF-35 Stimulates MIP3-a In vitro as shown in FIG. 9.

Example 11 LPF-35 in a Phenylquinone (PQ) Writhing Model of Pain in Mice

In this experiment, vehicle (LPF-35 diluent buffer, pH 7.0, 10 mL/kg)and LPF-35 (1000 mg/kg) are administered orally to 2 groups of 3 ICRmice weighing 22+/−2 g one hour before PQ is injected intraperitoneallyat 2 mg/kg. The number of writhings observed over the subsequent 5-10minute period is recorded. Similarly, mice are pre-treated with vehicleor LPF-35 at the same doses as above twice daily for 3 days. Two groupsof 3 ICR mice weighing 22+/−2 g are used. Sixty minutes following thelast dose, PQ is injected intraperitoneally at 2 mg/kg. The number ofwrithings is recorded over the following 5-10 minutes. Reduction in thenumber of writhing indicates analgesic activity. Morphine is used as apositive control.

Example 12 LPF-35 in a Paw Flinch Model of Pain in Rats

In this experiment the formalin paw flinch rat model is used to test thepotential analgesic effect and dose response of oral LPF-35. Theformalin test is widely used as a peripheral inflammatory nociceptivetest. Briefly, rats are given a subplantar injection of 2% formalin intothe right hind paw in a volume of 50 microliters. The animals are thentransferred to a Persplex observation chamber where pain behavior isquantified by counting the incidents of spontaneous flinching of theinjected paw during two distinct periods of flinching activity; (0-10min after injection) and (10-30 min after injection). Reduction in thenumber of paw flinches indicates analgesic activity. To define the bestexperimental conditions for LPF-35, several doses are tested. Inaddition, LPF-35 is administered orally at several time points beforethe nociceptive test. Morphine is used as a positive control.

Example 13 LPF-35 in a Tail Flick Model of Pain in Rats

In this model the tail-flick test was used to assess the analgesicproperties of LPF-35. Rats were restrained with a cloth restrainer. Themiddle dorsal surface of tail was heated with a focused beam of radiantheat. To measure analgesic effect, the time required to elicit a flickof the tail was recorded. An increase in the time required to elicit atail flick indicates analgesic activity. Various doses of LPF-35 wereadministered at different time points prior to the test. Morphine wasused as a positive control. A single oral dose of LPF-35 increased thetail-flick response time to heat by 16% compared to control.

Example 14 LPF-35 in an Acetic Acid Writhing Model of Pain in Rats

Another model that was used to test the analgesic efficacy of LPF-35 wasthe acetic acid writhing test in rats. Rats were administered an i.p.injection of acetic acid (0.5%, 20 mL/kg). The number of writhesexhibited for each animal during the 10 minutes following administrationof acetic acid was recorded. Reduction in the number of writhingindicates analgesic activity. Various doses of LPF-35 were administeredat different time points prior to the test. Morphine was used as apositive control. Immediately after the test, the rats were sacrificedand ascites were collected for measurement of PGE2 contents. PGE2 isbelieved to be an important mediator of inflammatory nociception. Asingle oral dose of LPF-35 administered 60 minutes prior to the testreduced the number of writhings by 18% compared to control.

Example 15 Tolerance Development with LPF-35 Versus Morphine

Chronic administration of morphine is known to result in the developmentof tolerance and a loss of analgesic effect in the rat. To test whetherrats can become tolerant to LPF-35, use the tail-flick model of pain inrats. This test is the most widely used nociceptive assay to measure thedevelopment of opioid tolerance in the rat spinal cord. Intrathecalcatheters are implanted under anesthesia. Rats are shaved andsterilized. The posterior superficial neck muscles are separated and acatheter inserted through the occipital membrane, gently advancing itcaudally to the subarachnoid space. The external end of the catheter iscapped until the day of the experiment. Animals showing normal motorfunction 5 days after surgery are used. Under light anesthesia, the testcompound is injected at a volume of 5 microliters and flushed with 10microliters saline 15 minutes before the test. Osmotic minipumps,implanted subcutaneously on the back under anesthesia, are used tocontinuously infuse the test compound. Various doses of LPF-35 areadministered over a period of days to assess the development oftolerance. Morphine is used as a positive control.

Example 16 Enhancement of Morphine Analgesia with LPF-35

Co-administration of LPF-35 may result in a synergistic response thatcould allow the reduction in the dose of morphine used. To examinewhether morphine and LPF-35 act synergistically, the rat tail flickmodel is used to test various doses of morphine with and without LPF-35co-administration. Briefly, rats are restrained with a cloth restrainer.The middle dorsal surface of tail is heated with a focused beam ofradiant heat. To measure analgesic effect, the time required to elicit aflick of the tail is recorded. Various doses of LPF-35, with and withoutco-administration of a sub-effective dose of morphine, are used to testwhether LPF-35 can potentiate the effects of Morphine.

Example 17 LPF-35 Effects on the 1-Opioid Receptor

This experiment examines whether LPF-35's analgesic effect is mediatedvia the μ-opioid receptor. In this experiment the paw flinch model inrats is used to determine whether co-administration of naloxone (NLX)and D-Phe-Cys-Tyr-D-Trp-Om-Thr-Nh2 (CTOP), 2 specific μ-opioid receptorantagonists, abrogate the analgesic effect of LPF-35.

Example 18 LPF-35 Effects on West Nile Virus in Hamsters

In this experiment one group of hamsters receives oral placebo (rhLFvehicle: PBS solution) and 4 additional groups receive 4 doses of oralLPF-35 (10, 50, 200 or 500 mg/kg) once per day via oral gavage. Threedifferent regimens are tested: 3 days before virus inoculation, the sameday of infection (day 0), and 3 days after infection for a total of 3experiments. 14 days after injection of the virus, the two groups ofhamsters are inoculated intraperitoneally with 104 TCID50 of WNV strainNY385-99. After virus inoculation (day 0), the animals are bled dailyfor 6-7 days to measure their level of viremia and immune response. Thehamsters are observed for 20 additional days for signs of illness ordeath.

Example 19 LPF-35 Effects in a Mouse Model of Squamous Cell Cancer

The effect of orally administered LPF-35 on the development of 012subcutaneous tumors are examined and compared to the effect of LPF-35administered by an intravenous (i.v.) route. Human squamous cellcarcinoma (012) is used, employing 5×106 cell in 200 mL injected to theright flank of athymic nude mice. Eleven days post tumor injection, whentumor volumes measure ˜20 mm3, mice are divided in three groups (1placebo control and two LPF-35-treated) at 8 animals per group. Onegroup is treated with 5 mg/kg LPF-35 i.v. (50 mL injections) once a dayfor five days; a second group is treated with 25 mg/kg LPF-35 orally bygavage (0.2 mL per dose) twice a day for eight days; the placebo groupis treated following the oral schedule. Tumors are measured twice a weekfor the duration of the experiment and the body weights are determinedat the time of tumor measurements.

Example 20 LPF-35 Effects in an Animal Model of Obesity

LPF-35 is tested in a mouse model of obesity. Four-week-old-male miceare fed D12492i 60% kcal fat diet from Research Diets Inc. until theyreach 8 weeks of age (total time on diet is 4 weeks). Control mice arefed D12450Bi 10% kcal diet from Research Diets, Inc. Mice areindividually ear-notched and housed 4 per cage. All 24 mice are weighedat the start of the protocol and weekly thereafter. Serum is collectedand stored at −80o C from all 24 mice at the start and the end of thefeeding protocol. Diet is weighed twice daily to determine food intake.The 16 DIO mice are divided into groups of eight. Group 1 receives aplacebo by oral gavage daily starting on day 15 for 15 days. Group 2receives LPF-35 by oral gavage daily starting on day 15 for 15 days.Following the 4 week feeding protocol for each group, mice are weighedand serum is analyzed for changes relative to control.

Example 21 LPF-35 Effects in an Animal Model of Hypercholesterolemia

The effects of LPF-35 on cholesterol are tested in a mouse model ofdiet-induced hypercholesterolemia. Mice (n=5) are fed a high cholesteroldiet until serum lipid levels stabilize. Oral LPF-35 or placebo is thenadministered daily for 5 consecutive days. At the end of that periodserum lipids are again measured and changes from baseline recorded.

Example 22 LPF-35 Effects in an In Vitro Model of Osteoporosis

The effect of LPF-35 in osteoporosis is determined by testing osteoclastmotility in vitro. Osteoclasts are taken from new-born rats (n=3-6). Theosteoclast suspension is then applied to culture dish (20 min).Unattached cells are washed away and incubated for 2 hours. At time 0,cells are viewed by inverted phase-contrast microscope. LPF-35 isadministered to incubation medium at time 30 min. Every 5 min cellimages are taken using time-lapse video (90 min). Change in position andspread area of osteoclasts is measured to calculate an index ofosteoclast motility.

Example 23 LPF-35 Effects in Organ Transplant and Graft-Versus-HostDisease

FK506 and cyclosporine are principal treatments for organ transplant andhave also been shown to inhibit GVHD. The activity of LPF-35 incombination with FK506 was compared with FK506 alone in a heartallograft survival model in rats. Wistar Furh (WF; RT1) recipients ofheart allograft from Baffalo donors (BUF; RT1b) were administered oralLPF-35 (5 mg/kg) plus FK506 (0.8 mg/kg), FK506 alone or placebo. Theresults are presented as a mean±standard deviation (MST±SD).Significance between groups was calculated using Student T-test withp<0.05 considered as significant. In group # 1 control WF recipients ofBUF heart allografts (n=5) were untreated and heart allografts wererejected in an acute fashion within 6.4±0.5 days. In group # 2 WFrecipients of BUF heart allografts (n=10) were treated daily once (days0 to +14) with 0.8 mg/kg FK506 delivered by oral gavage. This protocolwas effective in prolonging the survival of heart allografts to a MST of22.3±6.7 days (p=0.0002). These results suggest that FK506 alone in thistherapeutic protocol is very effective in protecting allografts fromrejection. In group # 3 WF recipients of BUF heart allografts (n=6) weretreated daily once (days 0 to +14) with 0.8 mg/kg FK506 delivered byoral gavage in combination with LPF-35 daily for 14 days. This protocolwas effective in prolonging the survival of heart allografts to a MST of49.5±28 days (p=0.0077). The results showed that LPF-35 was effective inimproving the results produced by FK506 alone. An effect of reducingorgan transplant rejection is indicative of beneficial effects in GVHD.

Example 24 LPF-35 Effects in an Rat Model of Alzheimer's

The effects of LPF-35 in Alzheimer's is tested using the Fimbria FornixLesion Model in rats. Axotomy of the fimbria formix, a cholinergicpathway that goes from the septum to the hippocampus, leads to adecrease in hippocampal ChAT content, memory impairment and hippocampalreorganization (sprouting). This procedure induces a degenerativeprocess both at the hippocampal terminals and at the septal cellularbodies. This model mimics some of the changes observed in Alzheimer'spatients and thus has been proposed to be a useful test in search of atherapy that could prevent or slow down the cholinergic neurondegeneration occurring in Alzheimer's disease.

Male Sprague-Dawley rats are anesthetized with pentobarbital.Electrolytic lesion (1 mA, 40 sec) of the fimbria-formix is then doneunilaterally. Animals are left to recover for 10 days. Oral LPF-35 isadministered for 10 days, first administration starting 1 hour beforelesion. ChAT activity is then measured in the hippocampus. Additionally,the number of ChAT immunoreactive positive neurons is counted in theseptum.

Example 25 LPF-35 Effects in a Mouse Model of Multiple Sclerosis

To test whether LPF-35 is potentially effective in Multiple Sclerosis,the experimental autoimmune encephalomyelitis model in mice is used. Inthis experiment it will also be investigated whether anterior uveitis(AU), which often accompanies central nervous system (CNS) and systemicinflammatory diseases including multiple sclerosis (MS), also developsin this murine relapsing model of MS.

Experimental autoimmune encephalomyelitis (EAE) closely resemblingrelapsing-remitting MS, is induced in the mice by immunization withmyelin basic protein (MBP). F1 female mice are immunized with MBP inComplete Freund's Adjuvant (CFA) using Pertussis toxin as co-adjuvant.LPF-35 or placebo is then administered orally for five days. Followingthe treatment period, EAE is scored clinically on a scale of 0-5 basedon the degree of paralysis observed in the mice. Uveitis is assessed byslit-lamp biomicroscopy. Histological analysis of the CNS and eye isalso performed.

Example 26 LPF-35 Effects in a Rat Model of Irritable Bowel Syndrome

The effects of LPF-35 on symptoms of Irritable Bowel Syndrome are testedusing a rat model of post-inflammatory visceral hyperalgesia.Administering an inflammatory compound to the gastro-intestinal tract isknown to produce symptoms similar to IBS including reduced G.I. motilityand colonic compliance. After stimulating G.I inflammation, Oral LPF-35or placebo is administered once a day for 5 days. G.I. motility andcompliance is measured and compared to baseline. Afterwards, animals aresacrificed and histopathology of the gut performed.

Example 27 LPF-35 Effects in an Animal Model of Crohn's Disease

This experiment the effects of LPF-35 are tested in an immunologic mousemodel of Crohn's Disease. Immunologically mediated models are defined asmodels of adoptively transferred T cells or bone marrow precursors,which are introduced into immunodeficient mice. TH1 polarization mayplay a key role in the pathogenesis of Crohn's. This model is used toinduce a TH1 immune response in mice that is then assessed with andwithout treatment with orally administered LPF-35. T cell response aremeasured along with serum cytokines to determine whether LPF-35 canalter the immunologic pathogenesis associated with Crohn's disease.

Example 28 LPF-35 Effects in a Rat Model of Endometriosis

This experiment LPF-35 is tested in a rat model of endometriosis. Theprimary method used for induction of endometriosis in rats has beenautotransplantation of uterine squares (implants) into the peritonealcavity. Beyond mere growth of endometrium in ectopic locations, ratswith endometriosis display similar symptoms, including a reduction infertility and fecundity, and the endometriotic implants react similarlyto therapeutics as those of humans with the disease. Using thistechnique it is determined whether the administration of oral LPF-35 hasan effect on the development of lesions in the endometrium and reducesthe other symptoms of endometriosis.

Example 29 LPF-35 Effects in a Mouse Model of Parkinson's Disease

LPF-35 is tested in a mouse model of Parkinson's Disease by using MPTPto test for changes in Parkinson's associated neurochemicals. MPTP isknown to cause an irreversible destruction of the dopaminergicnigrostriatal pathway and symptoms of parkinsonism in humans and inmonkeys. When MPTP is administered to mice, a decrease in concentrationsof striatal dopamine (DA) and its metabolites DOPAC and HVA and adisappearance of nerve cells in the zona compacta of the substantianigra are observed. Thus, MPTP administration causes biochemical andhistological changes in mice similar to those seen in Parkinson'sdisease. This model allows the determination of the potentialneuroprotective properties of a test compound on dopaminergic neuronsafter MPTP damage.

On day 1 MPTP (15 mg/kg, s.c.) is injected into male C57/BL6 mice oncedaily for 7 days. Oral LPF-35 is then administered daily for 14 days. Onday 28 striata are removed, homogenized in perchloric acid, andcentrifuged. The supernatant is removed and stored at −80° C. Dopamine,DOPAC, HVA, NA, 5-HT, 5-HIAA are determined by reverse-phase HPLC andelectrochemical detection.

Example 30 LPF-35 Effects in an Animal Model of Periodontal Disease

To test the effects of LPF-35 in periodontal disease, P/E-selectinadhesion molecule deficient mice that mimic the human syndrome leukocyteadhesion deficiency ere used. In comparison to wild type animals, P/E−/−mice exhibit a 10-fold elevation in bacterial colonization of their oralcavities and elevated gingival tissue levels of the bone resorptivecytokine IL-1alpha. Mice are administered oral LPF-35 or placebo for 14days. Subsequently, bacterial colonization of the oral cavities andextent of gingival tissue are measured and compared to control groups.

Example 31 LPF-35 Effects in a Mouse Model of Depression

This test creates a condition in which exposure to inescapable footshocks, an uncontrolled aversive stimulus, leads to a decreased abilityto escape future aversive situations. Antidepressants such as monoamineoxidase inhibitors, 5-HT1A agonists, selective 5-HT uptake inhibitors,imipramine or clomipramine have been shown to restore, at leastpartially, the ability of animals to escape these aversive situations.

Male Wistar rats are used in the following paradigm. For Phase 1:Helplessness induction: Inescapable electric foot shocks (0.8 mA, 10 secduration every 20 sec for 60 min) are administered. For Phase 2.Conditioned avoidance training: Avoidance training is performed in a2-way shuttle box on days 3, 4 and 5; 5 min habituation followed by 30avoidance trials; At 3 seconds light signal, animal can escape to safeside of box; 0.55 mA shock is delivered if animals fail to escape;Failure—animal fails to escape during second shock. Oral LPF-35 isadministered for 5 days followed by the helplessness induction test.

Example 32 LPF-35 Effects in an Animal Model of Sleep Disorder

Hexobarbital potentiation in mice (n=3) is used to determine the effectsof LPF-35 on sleep time in mice. Oral LPF-35 is administered once perday for five days prior to the test. On the test day hexobarbital sodium(90 mg/kg i.p.) is administered to induce sleep. Sleep time is thenrecorded. Sleep time is measured in each animal as time (maximum 180min) from loss to recovery of righting reflex.

Example 33 LPF-35 Effects in a Rat Model of Conjunctivitis

The aim of this study is to determine whether the presence oflymphocytic infiltrates observed in the histology of ocular allergicconditions in humans or in the late phase of ocular anaphylacticreactions in experimental animals is a non-specific event dependent onlyon the degranulation of mast cells, or is conditioned by a specificresponse to antigen. With this in mind, responses to antigen and toLPF-35 are compared in an experimental model of allergic conjunctivitis.Rats are sensitised to ovalbumin and challenged topically in the leftconjunctival sac either with ovalbumin or LPF-35. The presence of Tcells and activated T cells in the infiltrate is studied byimmunohistochemical staining on conjunctival tissue obtained at 4, 24,and 48 hours after challenge. Numbers of T cells in the conjunctivalinfiltrate are compared with LPF-35 challenged rats at 48 hours and withcontrols at 4, 24, and 48 hours.

Example 34 LPF-35 Effects in a Mouse Model of Candida Albicans

Specific-pathogen-free inbred CBA/N female mice 8 weeks old areintraperitoneally challenged with 5×10⁸ or 1×10⁹ blastoconidia. From theday of the challenge, LPF-35 or saline is injected intravenously for 5days. Survival at day ten is measured as the primary endpoint. Five miceare used in each treatment.

Example 35 LPF-35 Effects in an Animal Model of Lupus

An active chromatin-induced systemic lupus erythematosus (SLE)-likemouse model shown to be similar to idiopathic SLE in humans, is used.

Female BALB/c mice 6-week-old are immunized with 100 μg active orresting chromatin, isolated from ConA-actived syngeneicspleno-lymphocytes, in 200 μL Freund's complete adjuvant (containing BCG10 g/L) on d 0 and then boosted in Freund's incomplete adjuvant on d 14and chromatin suspension on d 28. Mice are treated with LPF-35 peptideby oral gavage, once-a-day at a dose range from 5 to 20 mg/kg, from day30 until the end of experiment. Mice are sacrificed on d 45 or d 60.

Plasma samples of mice are tested by enzyme-linked immunosorbent assays(ELISA) for the presence of IgG anti-dsDNA, -ssDNA, and anti-histoneantibodies. Sera are tested by ELISA for tumor necrosis factor-a(TNF-a). Spleno-lymphocyte proliferation assays is carried out and thelevels of interferon-g (IFN-g) in supernatants are measured. Proteinuriais measured. Kidneys are examined by direct immunohistochemical methodand light microscopy.

It is shown that the peptide attenuates the indicators of activechromatin-induced SLE-like disease in this mouse model of idiopathichuman SLE.

Example 36 LPF-35 Effects in an Animal Model of Rheumatoid Arthritis

A rat model of arthritis is used in which paw swelling is induced byinjecting Complete Freund's Adjuvant (CFA) (0.3 mg of killed M.tuberculosis in 0.1 mL of light mineral oil). The paw swelling ismeasured on days 1 and 5 (acute phase) and also on days 14 and 18(delayed phase). Additionally, changes in animal weight are alsorecorded over the 18 days of the experiment.

Typically, groups of 5 rats are used per each dosing condition. LPF-35peptide is administered orally once-a-day for 5 consecutive daysbeginning 1 h before CFA administration.

Administration of the peptide is expected to reduce the hind pawswelling compared to a vehicle-treated control animals.

Example 37 LPF-35 Effects in an Animal Model of Osteo Arthritis

The effects of peptides are examined using articular cartilage slicestreated with hyaluronan hexasaccharides (HA6). Full-thickness slices ofthe human articular cartilage (˜10×10×1 mm) are cultured directly in 1.0mL of medium containing 10% FBS. Following 2 days of culture forrecovery, the tissue slices are treated in the presence of HA6 (250μg/mL) with or without the peptide (LPF-35) in the presence of 5% offetal bovine serum (FBS). The concentration of LPF-35 peptide is in therange from 0.1 mg/mL to 1.0 mg/mL. The culture medium containing HA6with or without LPF-35 is changed every 4 days. Following 14 days ofincubation the slices are removed and embedded in O.C.T. embeddingcompound (Tissue-TekR, Electron Microscopy Sciences, Washington, Pa.).Cryostat sections (8.0 μm) are prepared and stained for Safranin-O andHA. Cartilage proteoglycan biosynthesis is examined by a (35)S-sulfateincorporation assay. Cartilage slices are also examined for accumulationof proteoglycan by Safranin-O, and hyaluronan by a specific biotinylatedprobe.

It is shown that treatment with the peptide results in an increasedsynthesis of cartilage proteoglycan especially retained in thecell-associated matrix. Co-treatment with the peptide inhibits theHA6-induced depletion of cell-associated matrices as well as HA6-induceddepletion of hyaluronan and proteoglycan within cartilage tissue slices.

These results demonstrate that treatment with the peptide can abrogatethe catabolic events associated with a HA6-induced matrix depletionmodel of osteoarthritis.

Example 38 Protective Role of Lactoferrin Derived Peptides in a MouseModel of Colitis

A WT strain of C. rodentium is used. Before infection, C. rodentium aregrown in Luria broth overnight, washed, and re-suspended in PBS.Bacteria concentration is determined by OD and confirmed by serialdilution and culture. WT and iNOS−/− C57BL/6 eight-week-old mice aregavaged with C. rodentium in 100 μl of PBS or PBS vehicle alone, at theinoculum concentration of 5×108 bacteria/mouse to produce consistentinduction of colitis. Mice are treated with peptides starting day 1postinfection, at pH 7 in the drinking water. Daily weights are obtainedfor each mouse. Water consumption is measured to determine the peptideintake for individual animals. Mice are sacrificed after 14 days or whenmoribund. Blood samples are obtained by intracardiac puncture, andcolons are collected, weighed, and divided. Tissues fixed in 10%buffered formalin are used for histology.

Sections (6 μm) are cut from paraffin sections and stained with H&E.Tissues are examined in a blinded manner by a pathologist (C.B.D.).Acute (neutrophilic) and chronic (lymphocytic) inflammation, andepithelial regenerative changes are each scored on a 0-4 scale, and thesum is used as an index of histologic injury.

Serum is deproteinized with an equal volume of 6% 5-sulfosalicylic acid.Concentrations of total reactive nitrogen metabolites are determined inthe serum, by HPLC analysis and chemiluminescence with an NO analyzer.

Histology and serum analysis suggest a protective effect of peptides inthis model of colitis.

REFERENCES CITED

All patents and publications mentioned in the specifications areindicative of the levels of those skilled in the art to which theinvention pertains. All patents and publications are herein incorporatedby reference to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by reference.

-   U.S. Pat. No. 5,571,691-   U.S. Pat. No. 5,571,697-   U.S. Pat. No. 5,571,896-   U.S. Pat. No. 5,629,001-   U.S. Pat. No. 6,080,559-   U.S. Pat. No. 5,919,913-   U.S. Pat. No. 6,228,614-   U.S. Pat. No. 6,455,687-   U.S. Pat. No. 6,277,817-   U.S. Pat. No. 6,066,469-   U.S. Pat. No. 6,100,054-   U.S. Pat. No. 6,333,311

Although the present invention and its advantages have been described indetail, it should be understood that various changes, substitutions andalterations can be made herein without departing from the invention asdefined by the appended claims. Moreover, the scope of the presentapplication is not intended to be limited to the particular embodimentsof the process, machine, manufacture, composition of matter, means,methods and steps described in the specification. As one will readilyappreciate from the disclosure, processes, machines, manufacture,compositions of matter, means, methods, or steps, presently existing orlater to be developed that perform substantially the same function orachieve substantially the same result as the corresponding embodimentsdescribed herein may be utilized. Accordingly, the appended claims areintended to include within their scope such processes, machines,manufacture, compositions of matter, means, methods, or steps.

1. A pharmaceutical composition comprising a lactoferrin related peptidethat induces modulation of the immune system whereby the compositionstimulates production of macrophage inflammatory protein-3alpha fromhepatocytes.
 2. The pharmaceutical composition of claim 1, wherein saidcomposition inhibits bacterial growth as measured by minimum inhibitoryconcentration (MIC).
 3. The pharmaceutical composition of claim 1,wherein said lactoferrin related peptide comprises at least fourconsecutive positively charged residues beginning at the N-terminus. 4.The pharmaceutical composition of claim 1, wherein said lactoferrinrelated peptide comprises at least four positively charged residuesbeginning at the C-terminus.
 5. The pharmaceutical composition of claim1, wherein said lactoferrin related peptide comprises at least fourpositively charged residues beginning at each of the N- and C-termini.6. The pharmaceutical composition of claim 1, wherein said lactoferrinrelated peptide comprises at least 25% positively charged residues. 7.The pharmaceutical composition of claim 1, wherein said lactoferrinrelated peptide comprises at least 33% positively charged residues. 8.The pharmaceutical composition of claim 6, wherein the positivelycharged residues are arginine, lysine or modified and unusual aminoacids.
 9. The pharmaceutical composition of claim 7, wherein thepositively charged residues are arginine, lysine or modified and unusualamino acids.
 10. The pharmaceutical composition of claim 1, furthercomprising a metal chelator.
 11. The pharmaceutical composition of claim10, wherein the metal chelator is selected from the group consisting ofethylenediaminetetraacetic acid (EDTA),[ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA),1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA), andhydroxyethlene triamine diacetic acid (HEDTA).
 12. The pharmaceuticalcomposition of claim 1, wherein said composition comprises an amino acidsequence of SEQ. ID. NO.1, SEQ. ID. NO.2, SEQ. ID. NO.3, SEQ. ID. NO.4,SEQ. ID. NO.5, SEQ. ID. NO.6, SEQ. ID. NO.7, SEQ. ID. NO.8, SEQ. ID.NO.9, SEQ. ID. NO. 10, or SEQ. ID. NO.11.
 13. An isolated polypeptidehaving an amino acid sequence as defined in SEQ. ID. NO. 1, SEQ. ID.NO.2, SEQ. ID. NO.3, SEQ. ID. NO.4, SEQ. ID. NO.5, SEQ. ID. NO.6, SEQ.ID. NO.7, SEQ. ID. NO.8, SEQ. ID. NO.9, SEQ. ID. NO.10, or SEQ. ID.NO.11.
 14. The isolated polypeptide of claim 13, wherein saidpolypeptide is admixed with a pharmaceutically acceptable carrier.15.-47. (canceled)